Cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof

ABSTRACT

An improved cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof. The iCP-SOCS3 recombinant protein may be used as protein-based anti-angiogenic agent by utilizing the platform technology for macromolecule intracellular transduction. The iCP-SOCS3 recombinant protein includes a SOCS3 protein and at least one advanced macromolecule transduction domain (aMTD)(s). The aMTD(s) is composed of 9-13 amino acid residues and has the following features (a)-(c): (a) having 3 or more amino acid residues independently selected from the group consisting of Ala, Val, Ile, Leu, and Pro; (b) having Proline as an amino acid residue corresponding to any one or more of positions 5, 7 and 8, and position 12 of its amino acid sequence; and (c) having an instability index of 40-60, an aliphatic index of 180-220, and a grand average of hydropathy (GRAVY) score of 2.1-2.6, according to Protparam computational formula.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Continuation-in-Part of U.S. patent application Ser. No.14/838,295 (pending) filed Aug. 27, 2015, claiming priority based onU.S. Provisional Patent Application No. 62/042,493 filed Aug. 27, 2014,the contents of all of which are incorporated herein by reference intheir entirety.

TECHNICAL FIELD

The present invention relates to providing improved cell-permeable(iCP)-SOCS3 recombinant protein and uses thereof. Preferably, theiCP-SOCS3 recombinant protein may be used as protein-basedanti-angiogenic agent by utilizing the platform technology formacromolecule intracellular transduction.

BACKGROUND ART

Tumor cells have the ability to spread to adjacent or distant organs,penetrate blood or lymphatic vessels, circulate through theintravascular stream, and then proliferate at another site: metastasis.For the metastatic spread of cancer tissue, growth of the vascularnetwork is important. The processes whereby new blood and lymphaticvessels form are called angiogenesis, resulting in the excessiveproliferation of cancer cells through the formation of a new vascularnetwork to supply nutrients, oxygen and immune cells and also to removewaste products.

Cytokines including IL-6 and interferon-gamma (IFN-γ) activate the Januskinase (JAK)/signal transducers and activators of transcription (STAT)signaling pathway, a vital role promoting the inflammation,carcinogenesis and angiogenesis. Cytokine signaling is strictlyregulated by the SOCS family proteins induced by different classes ofagonists, including cytokines, hormones and infectious agents. Amongthem, SOCS1 and SOCS3 are relatively specific to STAT1 and STAT3,respectively. SOCS1 inhibits JAK activation through its N-terminalkinase inhibitory region (KIR) by the direct binding to the activationloop of JAKs, while SOCS3 binds to janus kinases (JAKs)-proximal siteson the receptor through its SH2 domain and inhibits JAK activity thatblocks recruitment of STAT3. Both SOCS1 and SOCS3 promoteanti-inflammatory effects due to the suppression ofinflammation-inducing cytokine signaling. Furthermore, the SOCS box,another domain in SOCS proteins, interacts with E3 ubiquitin ligasesand/or couples the SH2 domain-binding proteins to theubiquitin-proteasome pathway. Therefore, SOCSs inhibit cytokinesignaling by suppressing JAK kinase activity and degrading the activatedcytokine receptor complex.

In connection with SOCSs and angiogenesis, the SOCS3 gene has beenimplicated as an angiogenesis inhibitor in the cancer development.Previous studies have reported that SOCS3 is transiently induced byinflammatory mediators and inhibits cytoplasmic effectors such as theJAK/STAT kinases. In addition, SOCS3 deactivates tyrosine kinasereceptor signaling, including the IGF-1 receptor, resulting in thesuppression of apoptosis and vascular sprouting of the endothelial cells(ECs). This means that SOCS3 plays an important role in the negativeregulation of the JAK/STAT pathway and contributes to the suppression ofangiogenesis by regulating the angiogenic potentials of endothelialcells.

REFERENCES

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DISCLOSURE Technical Problem

In the previous study, recombinant SOCS3 proteins that contain acell-penetrating peptide (CPP), membrane-translocating motif (MTM) fromfibroblast growth factor (FGF)-4, has been reported to negativelycontrol JAK/STAT signaling. These recombinant SOCS3 proteins inhibitedSTAT phosphorylation, inflammatory cytokines production and MHC-IIexpression in cultured and primary macrophages. In addition, SOCS3 fusedto MTM protected mice challenged with a lethal dose of the SEBsuper-antigen, by suppressing apoptosis and hemorrhagic necrosis inmultiple organs. However, the SOCS3 proteins fused to FGF4-derived MTMdisplayed extremely low solubility, poor yields and relatively low cell-and tissue-permeability. Therefore, the MTM-fused SOCS3 proteins werenot suitable for further clinical development as therapeutic agents.

Technical Solution

For MITT, six critical factors (length, bending potential, instabilityindex, aliphatic index, GRAVY, amino acid composition) have beendetermined through analysis of baseline hydrophobic CPPs. Advancedmacromolecule transduction domain (aMTD), newly designed based on thesesix critical factors, could optimize cell-/tissue-permeability of SOCS3proteins that have a therapeutic effects and develop them asprotein-based drugs. Further, in order to increase solubility and yieldof recombinant protein, solubilization domains (SDs) additionally fusedto the aMTD-SOCS3 recombinant protein, thereby notably increased thesolubility and manufacturing yield of the recombinant protein.

In this application, aMTD/SD-fused iCP-SOCS3 recombinant proteins(iCP-SOCS3), much improved physicochemical characteristics (solubilityand yield) and functional activity (cell-/tissue-permeability) comparedwith the protein fused only to FGF-4-derived MTM. In addition, the newlydeveloped iCP-SOCS3 proteins have now been demonstrated to havetherapeutic application in inhibiting angiogenesis, exploiting theability of SOCS3 to suppress JAK/STAT signaling. The present applicationrepresents that macromolecule intracellular transduction technology(MITT) enabled by the new hydrophobic CPPs that are aMTD may providenovel protein therapy through SOCS3-intracellular protein replacementagainst tumor-mediated angiogenesis or disease associated with anangiogenic disorder. These findings suggest that restoration of SOCS3 byreplenishing the intracellular SOCS3 with iCP-SOCS3 protein creates anew paradigm for anti-cancer therapy or therapy of disease associatedwith an angiogenic disorder, and the intracellular protein replacementtherapy with the SOCS3 recombinant protein fused to the combination ofaMTD and SD pair may be useful to treat the disease associated with anangiogenic disorder.

One aspect disclosed in the present application provides an improvedCell-Permeable (iCP)-SOCS3 recombinant protein, which comprises a SOCS3protein; and at least one advanced macromolecule transduction domain(aMTD) being composed of 9-13 amino acid residues and having improvedcell and/or tissue permeability, wherein the aMTD is fused to one end orboth ends of the SOCS3 protein and has the following features of:

(a) being composed of 3 or more amino acid residues selected from thegroup consisting of Ala, Val, Ile, Leu, and Pro;

(b) having Proline as an amino acid residue corresponding to any one ormore of positions 5 to 8, and 12 of its amino acid sequence; and

(c) having an instability index of 40-60; an aliphatic index of 180-220;and a grand average of hydropathy (GRAVY) of 2.1-2.6, as measured byProtparam.

According to one embodiment, the iCP-SOCS3 recombinant protein furthercomprises one or more solubilization domain (SD)(s), and the aMTD(s),SOCS3 protein and SD(s) may be randomly fused to one another.

According to another embodiment, the aMTD may form α-Helix structure.According to still another embodiment, the aMTD may be composed of 12amino acid residues and represented by the following general formula:

wherein X(s) independently refer to Alanine (A), Valine (V), Leucine (L)or Isoleucine (I); and Proline (P) can be positioned in one of U(s)(either 5′, 6′, 7′ or 8′). The remaining U(s) are independently composedof A, V, L or I, P at the 12′ is Proline.

Another aspect disclosed in the present application provides aniCP-SOCS3 recombinant protein which is represented by any one of thefollowing structural formulae:

A-B-C, A-C-B, B-A-C, B-C-A, C-A-B, C-B-A, A-C-B-C and other possiblecombinations, wherein A is an advanced macromolecule transduction domain(aMTD) having improved cell and/or tissue permeability, B is a SOCS3protein, and C is a solubilization domain (SD); and the aMTD is composedof 9-13 amino acid residues and has the following features of: (a) beingcomposed of 3 or more amino acids selected from the group consisting ofAla, Val, Ile, Leu, and Pro;

(b) having Proline as an amino acid residue corresponding to any one ormore of positions 5 to 8, and 12 of its amino acid sequence;

(c) having an instability index of 40-60; an aliphatic index of 180-220;and a grand average of hydropathy (GRAVY) of 2.1-2.6, as measured byProtparam; and

(d) forming α-Helix structure.

According to one embodiment disclosed in the present application, theSOCS3 protein may have an amino acid sequence of SEQ ID NO: 814.

According to another embodiment disclosed in the present application,the SOCS3 protein may be encoded by a polynucleotide sequence of SEQ IDNO: 815.

According to still another embodiment disclosed in the presentapplication, the SOCS3 protein may further include a ligand selectivelybinding to a receptor of a cell, a tissue, or an organ.

According to still another embodiment disclosed in the presentapplication, the at least one aMTD(s) may have an amino acid sequenceindependently selected from the group consisting of SEQ ID NOs: 1-240and 822.

According to still another embodiment disclosed in the presentapplication, the at least one aMTD(s) may be encoded by a polynucleotidesequence independently selected from the group consisting of SEQ ID NOs:241-480 and 823.

According to still another embodiment disclosed in the presentapplication, the one or more SD(s) may have an amino acid sequenceindependently selected from the group consisting of SEQ ID NOs: 798,799, 800, 801, 802, 803, and 804.

According to still another embodiment disclosed in the presentapplication, the one or more SD(s) may be encoded by a polynucleotidesequence independently selected from the group consisting of SEQ ID NOs:805, 806, 807, 808, 809, 810, and 811.

According to still another embodiment disclosed in the presentapplication, the iCP-SOCS3 recombinant protein may have a histidine-tagaffinity domain additionally fused to one end thereof.

According to still another embodiment disclosed in the presentapplication, the histidine-tag affinity domain may have an amino acidsequence of SEQ ID NO: 812.

According to still another embodiment disclosed in the presentapplication, the histidine-tag affinity domain may be encoded by apolynucleotide sequence of SEQ ID NO: 813.

According to still another embodiment disclosed in the presentapplication, the fusion may be formed via a peptide bond or a chemicalbond.

According to still another embodiment disclosed in the presentapplication, the iCP-SOCS3 recombinant protein may be used for thetreatment or prevention of cancers or diseases associated with anangiogenic disorder.

Still another aspect disclosed in the present application provides apolynucleotide sequence encoding the iCP-SOCS3 recombinant protein.

Still another aspect disclosed in the present application provides arecombinant expression vector including the polynucleotide sequence.

Still another aspect disclosed in the present application provides atransformant transformed with the recombinant expression vector.

Still another aspect disclosed in the present application provides apreparing method of the iCP-SOCS3 recombinant protein includingpreparing the recombinant expression vector; preparing the transformantusing the recombinant expression vector; culturing the transformant; andrecovering the recombinant protein expressed by the culturing.

Still another aspect disclosed in the present application provides acomposition including the iCP-SOCS3 recombinant protein as an activeingredient.

Still another aspect disclosed in the present application provides apharmaceutical composition for treating or preventing cancers ordiseases associated with an angiogenic disorder including the iCP-SOCS3recombinant protein as an active ingredient; and a pharmaceuticallyacceptable carrier.

Still another aspect disclosed in the present application provides useof the iCP-SOCS3 recombinant protein as a medicament for treating orpreventing cancers or diseases associated with an angiogenic disorder.

Still another aspect disclosed in the present application provides amedicament including the iCP-SOCS3 recombinant protein.

Still another aspect disclosed in the present application provides useof the iCP-SOCS3 recombinant protein in the preparation of a medicamentfor treating or preventing cancers or diseases associated with anangiogenic disorder.

Still another aspect disclosed in the present application provides amethod of treating or preventing cancers or diseases associated with anangiogenic disorder in a subject, the method including identifying asubject in need of treating or preventing cancers or diseases associatedwith an angiogenic disorder; and administering to the subject atherapeutically effective amount of the iCP-SOCS3 recombinant protein.

According to one embodiment disclosed in the present application, thesubject may be a mammal.

Unless defined otherwise, all terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thepresent invention belongs. Although a certain method and a material isdescribed herein, it should not be construed as being limited thereto,any similar or equivalent method and material to those may also be usedin the practice or testing of the present invention. All publicationsmentioned herein are incorporated herein by reference in their entiretyto disclose and describe the methods and/or materials in connection withwhich the publications are cited. It must be noted that as used hereinand in the appended claims, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.

A “peptide,” as used herein, refers to a chain-type polymer formed byamino acid residues which are linked to each other via peptide bonds,and used interchangeably with “polypeptide.” Further, a “polypeptide”includes a peptide and a protein.

Further, the term “peptide” includes amino acid sequences that areconservative variations of those peptides specifically exemplifiedherein. The term “conservative variation,” as used herein, denotes thereplacement of an amino acid residue by another, biologically similarresidue. Examples of conservative variations include substitution of onehydrophobic residue, such as isoleucine, valine, leucine, alanine,cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine, or methionine for another, or substitution of one polar residuefor another, for example, substitution of arginine for lysine, glutamicacid for aspartic acid, or glutamine for asparagine, and the like.Neutral hydrophilic amino acids which may be substituted for one anotherinclude asparagine, glutamine, serine, and threonine.

The term “conservative variation” also includes use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide. Such conservative substitutions arewithin the definition of the classes of the peptides disclosed in thepresent application.

A person having ordinary skill in the art may make similar substitutionsto obtain peptides having higher cell permeability and a broader hostrange. For example, one aspect disclosed in the present applicationprovides peptides corresponding to amino acid sequences (e.g. SEQ IDNOs: 1 to 240 and 822) provided herein, as well as analogues, homologs,isomers, derivatives, amidated variations, and conservative variationsthereof, as long as the cell permeability of the peptide remains.

Minor modifications to primary amino acid sequence disclosed in thepresent application may result in peptides which have substantiallyequivalent or enhanced cell permeability, as compared to the specificpeptides described herein. Such modifications may be deliberate, as bysite-directed mutagenesis, or may be spontaneous.

All peptides may be synthesized using L-amino acids, but D forms of allof the peptides may be synthetically produced. In addition, C-terminalderivatives, such as C-terminal methyl esters and C-terminal amidates,may be produced in order to increase the cell permeability of thepeptide according to one embodiment disclosed in the presentapplication.

All of the peptides produced by these modifications are included herein,as long as in the case of amidated versions of the peptide, the cellpermeability of the original peptide is altered or enhanced such thatthe amidated peptide is therapeutically useful. It is envisioned thatsuch modifications are useful for altering or enhancing cellpermeability of a particular peptide.

Furthermore, deletion of one or more amino acids may also result in amodification to the structure of the resultant molecule without anysignificant change in its cell permeability. This may lead to thedevelopment of a smaller active molecule which may also have utility.For example, amino- or carboxyl-terminal amino acids which may not berequired for the cell permeability of a particular peptide may beremoved.

The term “gene” refers to an arbitrary nucleic acid sequence or a partthereof having a functional role in protein coding or transcription, orregulation of other gene expression. The gene may be composed of allnucleic acids encoding a functional protein or a part of the nucleicacid encoding or expressing the protein. The nucleic acid sequence mayinclude a gene mutation in exon, intron, initiation or terminationregion, promoter sequence, other regulatory sequence, or a uniquesequence adjacent to the gene.

The term “primer” refers to an oligonucleotide sequence that hybridizesto a complementary RNA or DNA target polynucleotide and serves as thestarting points for the stepwise synthesis of a polynucleotide frommononucleotides by the action of a nucleotidyltransferase as occurs, forexample, in a polymerase chain reaction.

The term “coding region” or “coding sequence” refers to a nucleic acidsequence, a complement thereof, or a part thereof which encodes aparticular gene product or a fragment thereof for which expression isdesired, according to the normal base pairing and codon usagerelationships. Coding sequences include exons in genomic DNA or immatureprimary RNA transcripts, which are joined together by the cellularbiochemical machinery to provide a mature mRNA. The anti-sense strand isthe complement of the nucleic acid, and the coding sequence may bededuced therefrom.

One aspect disclosed in the present application provides an iCP-SOCS3recombinant protein, which comprises a SOCS3 protein and at least oneadvanced macromolecule transduction domain (aMTD)(s) being composed of9-13 amino acid residues, preferably 10-12 amino acid residues, andhaving improved cell and/or tissue permeability, wherein the aMTD isfused to one end or both ends of the SOCS3 protein and has the followingfeatures of:

(a) being preferably composed of 3 or more amino acid residues selectedfrom the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having Proline as an amino acid residue corresponding to any one ormore of positions 5 to 8, and 12 of its amino acid sequence, andpreferably one or more of positions 5 to 8 and position 12 of its aminoacid sequence; and

(c) having an instability index of preferably 40-60 and more preferably41-58; an aliphatic index of preferably 180-220 and more preferably185-225; and a grand average of hydropathy (GRAVY) of preferably 2.1-2.6and more preferably 2.2-2.6 as measured by Protparam (seeweb.expasy.org).

To determine the GRAVY value of the protein analysed easily, theProtParam (Gasteiger E. et al., Protein Identification and AnalysisTools on the ExPASy Server, J M Walker ed., The Proteomics ProtocolsHandbook, Humana Press, 2005, 571-607) program is used. ProtParamprogram (web.expasy.org/protparam/) is a computational formula whichprovides various physicochemical properties of the proteins studied byanalysing their sequence; when the said amino-acid sequence is entered,the program calculates the GRAVY value of the protein whose degree ofhydrophobicity is to be measured.

These critical factors that facilitate the cell permeable ability ofaMTD sequences were analyzed, identified, and determined according toone embodiment disclosed in the present application. These aMTDsequences are artificially assembled based on the critical factors (CFs)determined from in-depth analysis of previously published hydrophobicCPPs.

The aMTD sequences according to one aspect disclosed in the presentapplication are the first artificially developed cell permeablepolypeptides capable of mediating the transduction of biologicallyactive macromolecules—including peptides, polypeptides, protein domains,or full-length proteins—through the plasma membrane of cells.

According to one embodiment, the iCP-SOCS3 recombinant protein furthercomprises one or more solubilization domain (SD)(s), and the aMTD(s),SOCS3 protein and SD(s) may be randomly fused to one another. Forexample, SD(s) may be further fused to one or more of the SOCS3 proteinand the aMTD, preferably one end or both ends of the SOCS3 protein, andmore preferably to the C-terminus of the SOCS3 protein.

According to another embodiment, the aMTD may form α-Helix structure.

According to still another embodiment, the aMTD may be preferablycomposed of 12 amino acid residues and represented by the followinggeneral formula:

Here, X(s) independently refer to Alanine (A), Valine (V), Leucine (L)or Isoleucine (I); and Proline (P) can be positioned in one of U(s)(either 5′, 6′, 7′ or 8′). The remaining U(s) are independently composedof A, V, L or I, P at the 12′ is Proline.

Still another aspect disclosed in the present application provides aniCP-SOCS3 recombinant protein which is represented by any one ofstructural formulae A-B-C, A-C-B, B-A-C, B-C-A, C-A-B, C-B-A, A-C-B-Cand other possible combinations, preferably by A-B-C or C-B-A:

wherein A is an advanced macromolecule transduction domain (aMTD) havingimproved cell and/or tissue permeability, and if the iCP-SOCS3recombinant protein comprises two or more aMTDs, they can be same ordifferent; B is a SOCS3 protein; and C is a solubilization domain (SD),and if the iCP-SOCS3 recombinant protein comprises two or more SDs, theycan be same or different; and the aMTD is composed of 9-13, preferably10-12 amino acid residues and has the following features of:

(a) being composed of 3 or more amino acid residues selected from thegroup consisting of Ala, Val, Ile, Leu, and Pro;

(b) having Proline as an amino acid residue corresponding to any one ormore of positions 5 to 8, and 12 of its amino acid sequence, andpreferably, one or more of positions 5 to 8 and position 12 of its aminoacid sequence;

(c) having an instability index of 40-60, preferably 41-58 and morepreferably 50-58; an aliphatic index of 180-220. preferably 185-225 andmore preferably 195-205; and a grand average of hydropathy (GRAVY) of2.1-2.6 and preferably 2.2-2.6, as measured by Protparam (seeweb.expasy.org); and

(d) preferably forming α-Helix structure.

In one embodiment disclosed in the present application, the SOCS3protein may have an amino acid sequence of SEQ ID NO: 814.

In another embodiment disclosed in the present application, the SOCS3protein may be encoded by a polynucleotide sequence of SEQ ID NO: 815.

When the iCP-SOCS3 recombinant protein is intended to be delivered to aparticular cell, tissue, or organ, the SOCS3 protein may form a fusionproduct, together with an extracellular domain of a ligand capable ofselectively binding to a receptor which is specifically expressed on theparticular cell, tissue, or organ, or monoclonal antibody (mAb) capableof specifically binding to the receptor or the ligand and a modifiedform thereof.

The binding of the peptide and a biologically active substance may beformed either by indirect linkage by a cloning technique using anexpression vector at a nucleotide level or by direct linkage viachemical or physical covalent or non-covalent bond of the peptide andthe biologically active substance.

In still another embodiment disclosed in the present application, theSOCS3 protein may preferably further include a ligand selectivelybinding to a receptor of a cell, a tissue, or an organ.

In one embodiment disclosed in the present application, the at least oneaMTD(s) may have an amino acid sequence independently selected from thegroup consisting of SEQ ID NOs: 1-240 and 822, preferably SEQ ID NOs: 2,16, 22, 32, 40, 43, 63, 65, 77, 84, 85, 86, 110, 131, 142, 143, 177,228, 229, 233, 237, 239 and 822, more preferably SEQ ID NO: 43.

In still another embodiment disclosed in the present application, the atleast one aMTD(s) may be encoded by a polynucleotide sequenceindependently selected from the group consisting of SEQ ID NOs: 241-480and 823, preferably SEQ ID NOs: 242, 256, 262, 272, 280, 283, 303, 305,317, 324, 325, 326, 350, 371, 382, 383, 417, 468, 469 473, 477, 479 and823, more preferably SEQ ID NO: 283.

In still another embodiment disclosed in the present application, theone or more SD(s) may have an amino acid sequence independently selectedfrom the group consisting of SEQ ID NOs: 798, 799, 800, 801, 802, 803,and 804. The SD may be preferably SDA of SEQ ID NO: 798, SDB of SEQ IDNO: 799, or SDB′ of SEQ ID NO: 804, and more preferably, SDB of SEQ IDNO: 799 which has superior structural stability, or SDB′ of SEQ ID NO:804 which has a modified amino acid sequence of SDB to avoid immuneresponses upon in vivo application. The modification of the amino acidsequence in SDB may be replacement of an amino acid residue, Valine,corresponding to position 28 of the amino acid sequence of SDB (SEQ IDNO: 799) by Leucine.

In still another embodiment disclosed in the present application, theone or more SDs may be encoded by a polynucleotide sequenceindependently selected from the group consisting of SEQ ID NOs: 805,806, 807, 808, 809, 810, and 811. The SD may be preferably SDA encodedby a polynucleotide sequence of SEQ ID NO: 805, SDB encoded by apolynucleotide sequence of SEQ ID NO: 806, or SDB′ for deimmunization(or humanization) encoded by a polynucleotide sequence of SEQ ID NO:811, and more preferably, SDB having superior structural stability,which is encoded by a polynucleotide sequence of SEQ ID NO: 806, or SDB′having a modified polynucleotide sequence of SDB to avoid immuneresponses upon in vivo application, which is encoded by a polynucleotidesequence of SEQ ID NO: 811.

In still another embodiment disclosed in the present application, theiCP-SOCS3 recombinant protein may be preferably selected from the groupconsisting of:

1) a recombinant protein, in which SOCS3 having an amino acid sequenceof SEQ ID NO: 814 is fused to the N-terminus or the C-terminus of aMTDhaving any one amino acid sequence selected from the group consisting ofSEQ ID NOs: 1 to 240 and 822, 2, 16, 22, 32, 40, 43, 63, 65, 77, 84, 85,86, 110, 131, 142, 143, 177, 228, 229, 233, 237, 239 and 822, morepreferably SEQ ID NO: 43;

2) a recombinant protein, in which SD having any one amino acid sequenceselected from the group consisting of SEQ ID NOs: 798 to 804 is furtherfused to one or more of the N-terminus or the C-terminus of the SOCS3and aMTD in the recombinant protein of 1); and

3) a recombinant protein, in which a Histidine tag having an amino acidsequence of 812 is further fused to the N-terminus of the recombinantprotein of 1) or 2).

According to one embodiment, the iCP-SOCS3 recombinant protein may becomposed of an amino acid sequence represented by SEQ ID NO: 825.

The SOCS3 protein may exhibit a physiological phenomenon-relatedactivity or a therapeutic purpose-related activity by intracellular orin-vivo delivery. The recombinant expression vector may include a tagsequence which makes it easy to purify the recombinant protein, forexample, consecutive histidine codon, maltose binding protein codon, Myccodon, etc., and further include a fusion partner to enhance solubilityof the recombinant protein, etc. Further, for the overall structural andfunctional stability of the recombinant protein or flexibility of theproteins encoded by respective genes, the recombinant expression vectormay further include one or more glycine, proline, and spacer amino acidor polynucleotide sequences including AAY amino acids. Furthermore, therecombinant expression vector may include a sequence specificallydigested by an enzyme in order to remove an unnecessary region of therecombinant protein, an expression regulatory sequence, and a marker orreporter gene sequence to verify intracellular delivery, but is notlimited thereto.

In still another embodiment disclosed in the present application, theiCP-SOCS3 recombinant protein may preferably have a histidine-tagaffinity domain additionally fused to one end thereof.

In still another embodiment disclosed in the present application, thehistidine-tag affinity domain may have an amino acid sequence of SEQ IDNO: 812.

In still another embodiment disclosed in the present application, thehistidine-tag affinity domain may be encoded by a polynucleotidesequence of SEQ ID NO: 813.

In still another embodiment disclosed in the present application, thefusion may be formed via a peptide bond or a chemical bond.

The chemical bond may be preferably selected from the group consistingof disulfide bonds, diamine bonds, sulfide-amine bonds, carboxyl-aminebonds, ester bonds, and covalent bonds.

In still another embodiment disclosed in the present application, theiCP-SOCS3 recombinant protein may be used for the treatment orprevention of cancers or diseases associated with an angiogenicdisorder.

Still another aspect disclosed in the present application provides apolynucleotide sequence encoding the iCP-SOCS3.

According to still another embodiment disclosed in the presentapplication, the polynucleotide sequence may be fused with ahistidine-tag affinity domain.

Still another aspect disclosed in the present application provides arecombinant expression vector including the polynucleotide sequence.

Preferably, the vector may be inserted in a host cell and recombinedwith the host cell genome, or refers to any nucleic acid including anucleotide sequence competent to replicate spontaneously as an episome.Such a vector may include a linear nucleic acid, a plasmid, a phagemid,a cosmid, an RNA vector, a viral vector, etc.

Preferably, the vector may be genetically engineered to incorporate thenucleic acid sequence encoding the recombinant protein in an orientationeither N-terminal and/or C-terminal to a nucleic acid sequence encodinga peptide, a polypeptide, a protein domain, or a full-length protein ofinterest, and in the correct reading frame so that the recombinantprotein consisting of aMTD, SOCS3 protein, and preferably SD may beexpressed. Expression vectors may be selected from those readilyavailable for use in prokaryotic or eukaryotic expression systems.

Standard recombinant nucleic acid methods may be used to express agenetically engineered recombinant protein. The nucleic acid sequenceencoding the recombinant protein according to one embodiment disclosedin the present application may be cloned into a nucleic acid expressionvector, e.g., with appropriate signal and processing sequences andregulatory sequences for transcription and translation, and the proteinmay be synthesized using automated organic synthetic methods. Syntheticmethods of producing proteins are described in, for example, theliterature [Methods in Enzymology, Volume 289: Solid-Phase PeptideSynthesis by Gregg B. Fields (Editor), Sidney P. Colowick, Melvin I.Simon (Editor), Academic Press (1997)].

In order to obtain high level expression of a cloned gene or nucleicacid, for example, a cDNA encoding the recombinant protein according toone embodiment disclosed in the present application, the recombinantprotein sequence may be typically subcloned into an expression vectorthat includes a strong promoter for directing transcription, atranscription/translation terminator, and in the case of a nucleic acidencoding a protein, a ribosome binding site for translationalinitiation. Suitable bacterial promoters are well known in the art andare described, e.g., in the literatures [Sambrook & Russell, MolecularCloning: A Laboratory Manual, 3d Edition, Cold Spring Harbor Laboratory,N.Y. (2001); and Ausubel, et al., Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley Interscience, N. Y.(1989)]. Bacterial expression systems for expression of the recombinantprotein are available in, e.g., E. coli, Bacillus sp., and Salmonella(Palva et al., Gene 22: 229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for such expression systems are commerciallyavailable. Eukaryotic expression systems for mammalian cells, yeast, andinsect cells are well known in the art and are also commerciallyavailable. The eukaryotic expression vector may be preferably anadenoviral vector, an adeno-associated vector, or a retroviral vector.

Generally, the expression vector for expressing the cell permeablerecombinant protein according to one embodiment disclosed in the presentapplication in which the cargo protein, i.e. ΔSOCS3 protein, is attachedto the N-terminus, C-terminus, or both termini of aMTD may includeregulatory sequences including, for example, a promoter, operablyattached to a sequence encoding the advanced macromolecule transductiondomain. Non-limiting examples of inducible promoters that may be usedinclude steroid-hormone responsive promoters (e.g., ecdysone-responsive,estrogen-responsive, and glutacorticoid-responsive promoters),tetracycline “Tet-On” and “Tet-Off” systems, and metal-responsivepromoters.

The polynucleotide sequence according to one embodiment disclosed in thepresent application may be present in a vector in which thepolynucleotide sequence is operably linked to regulatory sequencescapable of providing for the expression of the polynucleotide sequenceby a suitable host cell.

According to one embodiment disclosed in the present application, thepolynucleotide sequence may be selected from the following groups:

1) a polynucleotide sequence, in which any one polynucleotide sequenceselected from the group consisting of SEQ ID NOs: 241-480 and 823,preferably SEQ ID NOs: 242, 252, 274, 279, 322, 331, 338, 345, 347, 361,365, 370, 371, 383, 387, 417, 462, 468, 469, 473, 477, 479 and 823, morepreferably SEQ ID NO: 283, is operably linked with a polynucleotidesequence of SEQ ID NO: 815; and

2) a polynucleotide sequence, in which any one polynucleotide sequenceselected from the group consisting of SEQ ID NOs: 805 to 811 is furtheroperably linked to the polynucleotide sequence of 1), or furtheroperably linked to between: any one polynucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 241-480 and 823, preferably SEQID NOs: 242, 256, 262, 272, 280, 283, 303, 305, 317, 324, 325, 326, 350,371, 382, 383, 417, 468, 469, 473, 477, 479 and 823, more preferably SEQID NO: 283; and a polynucleotide sequence of SEQ ID NO: 815.

Within an expression vector, the term “operably linked” is intended tomean that the polynucleotide sequence of interest is linked to theregulatory sequence(s) in a manner which allows for expression of thepolynucleotide sequence. The term “regulatory sequence” is intended toinclude promoters, enhancers, and other expression control elements.Such operable linkage with the expression vector can be achieved byconventional gene recombination techniques known in the art, whilesite-directed DNA cleavage and linkage are carried out by usingconventional enzymes known in the art.

The expression vectors may contain a signal sequence or a leadersequence for membrane targeting or secretion, as well as regulatorysequences such as a promoter, an operator, an initiation codon, atermination codon, a polyadenylation signal, an enhancer and the like.The promoter may be a constitutive or an inducible promoter. Further,the expression vector may include one or more selectable marker genesfor selecting the host cell containing the expression vector, and mayfurther include a polynucleotide sequence that enables the vector toreplicate in the host cell in question.

The expression vector constructed according to one embodiment disclosedin the present application may be the vector where the polynucleotideencoding the iCP-SOCS3 recombinant protein (where an aMTD is fused tothe N-terminus or C-terminus of a SOCS3 protein) is inserted within themultiple cloning sites (MCS), preferably within the Nde1/Sal1 site orBamH1/Sal1 site of a pET-28a(+)(Novagen, Darmstadt, Germany) orpET-26b(+) vector (Novagen, Darmstadt, Germany).

In still another embodiment disclosed in the present application, thepolynucleotide encoding the SD being additionally fused to theN-terminus or C-terminus of a SOCS3 protein or an aMTD may be insertedinto a cleavage site of restriction enzyme (Nde1, BamH1 and Sal1, etc.)within the multiple cloning sites (MCS) of a pET-28a(+)(Novagen,Darmstadt, Germany) or pET-26b(+) vector (Novagen, Darmstadt, Germany).

In still another embodiment disclosed in the present application, thepolynucleotide encoding the iCP-SOCS3 recombinant protein may be clonedinto a pET-28a(+) vector bearing a His-tag sequence so as to fuse sixhistidine residues to the N-terminus of the iCP-SOCS3 recombinantprotein to allow easy purification.

According to one embodiment disclosed in the present application, thepolynucleotide sequence may be a polynucleotide sequence selected fromthe group consisting of SEQ ID NOS: 824, 826, 828 and 830.

The recombinant protein may be introduced into an appropriate host cell,e.g., a bacterial cell, a yeast cell, an insect cell, or a tissueculture cell. The recombinant protein may also be introduced intoembryonic stem cells in order to generate a transgenic organism. Largenumbers of suitable vectors and promoters are known to those skilled inthe art and are commercially available for generating the recombinantprotein.

Known methods may be used to construct vectors including thepolynucleotide sequence according to one embodiment disclosed in thepresent application and appropriate transcriptional/translationalcontrol signals. These methods include in vitro recombinant DNAtechniques, synthetic techniques, and in vivo recombination/geneticrecombination. For example, these techniques are described in theliteratures [Sambrook & Russell, Molecular Cloning: A Laboratory Manual,3d Edition, Cold Spring Harbor Laboratory, N. Y. (2001); and Ausubel etal., Current Protocols in Molecular Biology Greene Publishing Associatesand Wiley Interscience, N.Y. (1989)].

Still another aspect disclosed in the present application provides atransformant transformed with the recombinant expression vector.

The transformation includes transfection, and refers to a processwhereby a foreign (extracellular) DNA, with or without an accompanyingmaterial, enters into a host cell. The “transfected cell” refers to acell into which the foreign DNA is introduced into the cell, and thusthe cell harbors the foreign DNA. The DNA may be introduced into thecell so that a nucleic acid thereof may be integrated into thechromosome or replicable as an extrachromosomal element. The cellintroduced with the foreign DNA, etc. is called a transformant.

As used herein, ‘introducing’ of a protein, a peptide, an organiccompound into a cell may be used interchangeably with the expression of‘carrying,’ ‘penetrating,’ ‘transporting,’ ‘delivering,’ ‘permeating’ or‘passing.’

It is understood that the host cell refers to a eukaryotic orprokaryotic cell into which one or more DNAs or vectors are introduced,and refers not only to the particular subject cell but also to theprogeny or potential progeny thereof. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

The host cells may be preferably bacterial cells, and as the bacterialcells, there are, in principle, no limitations. They may be eubacteria(gram-positive or gram-negative) or archaebacteria, as long as theyallow genetic manipulation for insertion of a gene of interest,preferably for site-specific integration, and they may be cultured on amanufacturing scale. Preferably, the host cells may have the property toallow cultivation to high cell densities.

Examples of bacterial host cells that may be used in the preparation ofthe recombinant protein are E. coli (Lee, 1996; Hannig and Makrides,1998), Bacillus subtilis, Pseudomonas fluorescens (Squires et al., 2004;Retallack et al., 2006) as well as various Corynebacterium (US2006/0003404 A1) and Lactococcus lactis (Mierau et al., 2005) strains.Preferably, the host cells are Escherichia coli cells.

More preferably, the host cell may include an RNA polymerase capable ofbinding to a promoter regulating the gene of interest. The RNApolymerase may be endogenous or exogenous to the host cell.

Preferably, host cells with a foreign strong RNA polymerase may be used.For example, Escherichia coli strains engineered to carry a foreign RNApolymerase (e.g. like in the case of using a T7 promoter a T7-like RNApolymerase in the so-called “T7 strains”) integrated in their genome maybe used. Examples of T7 strains, e.g. BL21(DE3), HMS174(DE3), and theirderivatives or relatives (see Novagen, pET System manual, 11^(th)edition), may be widely used and commercially available. Preferably,BL21-CodonPlus (DE3)-RIL or BL21-CodonPlus (DE3)-RIPL (AgilentTechnologies) may be used. These strains are DE3 lysogens containing theT7 RNA polymerase gene under control of the lacUV5 promoter. Inductionwith IPTG allows production of T7 RNA polymerase which then directs theexpression of the gene of interest under the control of the T7 promoter.

The host cell strains, E. coli BL21(DE3) or HMS174(DE3), which havereceived their genome-based T7 RNA polymerase via the phage DE3, arelysogenic. It is preferred that the T7 RNA polymerase contained in thehost cell has been integrated by a method which avoids, or preferablyexcludes, the insertion of residual phage sequences in the host cellgenome since lysogenic strains have the disadvantage to potentiallyexhibit lytic properties, leading to undesirable phage release and celllysis.

Still another aspect disclosed in the present application provides apreparing method of the iCP-SOCS3 recombinant protein includingpreparing the recombinant expression vector; preparing the transformantusing the recombinant expression vector; culturing the transformant; andrecovering the recombinant protein expressed by culturing.

Culturing may be preferably in a mode that employs the addition of afeed medium, this mode being selected from the fed-batch mode,semi-continuous mode, or continuous mode, and the bacterial expressionhost cells may include a DNA construct, integrated in their genome,carrying the DNA sequence encoding the protein of interest under thecontrol of a promoter that enables expression of said protein.

There are no limitations in the type of the culture medium. The culturemedium may be semi-defined, i.e. containing complex media compounds(e.g. yeast extract, soy peptone, casamino acids), or it may bechemically defined, without any complex compounds. Preferably, a definedmedium may be used. The defined media (also called minimal or syntheticmedia) are exclusively composed of chemically defined substances, i.e.carbon sources such as glucose or glycerol, salts, vitamins, and, inview of a possible strain auxotrophy, specific amino acids or othersubstances such as thiamine. Most preferably, glucose may be used as acarbon source. Usually, the carbon source of the feed medium serves asthe growth-limiting component which controls the specific growth rate.

Host cells may be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or the use ofcell lysing agents. The literature [Scopes, Protein Purification:Principles and Practice, New York: Springer-Verlag (1994)] describes anumber of general methods for purifying recombinant (andnon-recombinant) proteins. The methods may include, e.g., ion-exchangechromatography, size-exclusion chromatography, affinity chromatography,selective precipitation, dialysis, and hydrophobic interactionchromatography. These methods may be adapted to devise a purificationstrategy for the cell permeable recombinant protein. If the cellpermeable recombinant protein includes a purification handle, such as anepitope tag or a metal chelating sequence, affinity chromatography maybe used to easily purify the protein.

The amount of the protein produced may be evaluated by detecting theadvanced macromolecule transduction domain directly (e.g., using Westernanalysis) or indirectly (e.g., by assaying materials derived from thecells for specific DNA binding activity, such as by electrophoreticmobility shift assay). Proteins may be detected prior to purification,during any stage of purification, or after purification. In someimplementations, purification or complete purification may not benecessary.

The iCP-SOCS3 recombinant proteins according to one embodiment disclosedin the present application are cell permeable proteins, and may be usedas protein-based vaccines, particularly in the case where killed orattenuated whole organism vaccines are impractical.

The iCP-SOCS3 recombinant proteins according to one embodiment disclosedin the present application may be preferably used for the treating orpreventing cancers or diseases associated with an angiogenic disorder.The cell permeable recombinant proteins may be delivered to the interiorof the cell, eliminating the need to transfect or transform the cellwith a recombinant vector. The cell permeable recombinant proteins maybe used in vitro to investigate protein function or may be used tomaintain cells in a desired state.

Still another aspect disclosed in the present application provides acomposition including the iCP-SOCS3 Recombinant Protein as an activeingredient.

Still another aspect disclosed in the present application provides apharmaceutical composition for treating or preventing cancers ordiseases associated with an angiogenic disorder including the iCP-SOCS3Recombinant Protein as an active ingredient; and a pharmaceuticallyacceptable carrier.

According to one embodiment disclosed in the present application, theiCP-SOCS3 Recombinant Protein may be used in a single agent, or incombination with one or more other anti-cancer agents and/oranti-angiogenic agents.

Diseases associated with an angiogenic disorder may include, but are notlimited to, angiogenesis of the eye associated with ocular disorderincluding retinopathy of prematurity, diabetic macular edema, diabeticretinopathy, age-related macular degeneration, glaucoma, retinitispigmentosa, cataract formation, retinoblastoma and retinal ischemia; orcancer, arthritis, hypertension, kidney disease, psoriasis, maculardegeneration, and the like.

Preferably, the composition may be for injectable (e.g. intraperitoneal,intravenous, and intra-arterial, etc.) and may include the activeingredient in an amount of 0.001 mg/kg to 1000 mg/kg, preferably 0.01mg/kg to 100 mg/kg, more preferably 0.1 mg/kg to 50 mg/kg for human.

For examples, dosages per day normally fall within the range of about0.001 to about 1000 mg/kg of body weight. In the treatment of adulthumans, the range of about 0.1 to about 50 mg/kg/day, in single ordivided dose, is especially preferred. However, it will be understoodthat the concentration of the iCP-SOCS3 recombinant protein actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the age, weight, and response of theindividual patient, and the severity of the patient's symptoms, andtherefore the above dosage ranges are not intended to limit the scope ofthe invention in any way. In some instances dosage levels below thelower limit of the aforesaid range may be more than adequate, while inother cases still larger doses may be employed without causing anyharmful side effect, provided that such larger doses are first dividedinto several smaller doses for administration throughout the day.

Still another aspect disclosed in the present application provides useof the iCP-SOCS3 recombinant protein as a medicament for treating orpreventing cancers or diseases associated with an angiogenic disorder.

Still another aspect disclosed in the present application provides amedicament including the iCP-SOCS3 recombinant protein.

Still another aspect disclosed in the present application provides useof the iCP-SOCS3 recombinant protein for the preparation of a medicamentfor treating or preventing cancers or diseases associated with anangiogenic disorder.

Still another aspect disclosed in the present application provides amethod of treating or preventing cancers or diseases associated with anangiogenic disorder in a subject including identifying a subject in needof treating or preventing cancers or diseases associated with anangiogenic disorder; and administering to the subject a therapeuticallyeffective amount of the iCP-SOCS3 recombinant protein.

In one embodiment disclosed in the present application, the subject maybe preferably a mammal.

Preferably, the subject in need of treating or preventing cancers ordiseases associated with an angiogenic disorder may be identified by anyconventional diagnostic methods known in the art including ultrasound,CT scan, MRI, alpha-fetoprotein testing, des-gamma carboxyprothrombinscreening, and biopsy, etc.

The pharmaceutical composition according to one embodiment disclosed inthe present application may be prepared by using pharmaceuticallysuitable and physiologically acceptable additives, in addition to theactive ingredient, and the additives may include excipients,disintegrants, sweeteners, binders, coating agents, blowing agents,lubricants, glidants, flavoring agents, etc.

For administration, the pharmaceutical composition may be preferablyformulated by further including one or more pharmaceutically acceptablecarriers in addition to the above-described active ingredient.

Dosage forms of the pharmaceutical composition may include granules,powders, tablets, coated tablets, capsules, suppositories, liquidformulations, syrups, juice, suspensions, emulsions, drops, injectableliquid formulations, etc. For formulation of the composition into atablet or capsule, for example, the active ingredient may be combinedwith any oral, non-toxic pharmaceutically acceptable inert carrier, suchas ethanol, glycerol, water, etc. If desired or necessary, suitablebinders, lubricants, disintegrants, and colorants may be additionallyincluded as a mixture.

Examples of the suitable binder may include, but are not limited to,starch, gelatin, natural sugars such as glucose or beta-lactose, cornsweeteners, natural and synthetic gums such as acacia, tragacanth, orsodium oleate, sodium stearate, magnesium stearate, sodium benzoate,sodium acetate, sodium chloride, etc. Examples of the disintegrant mayinclude, but are not limited to, starch, methyl cellulose, agar,bentonite, xanthan gum, etc. For formulation of the composition into aliquid preparation, a pharmaceutically acceptable carrier which issterile and biocompatible may be used, such as saline, sterile water, aRinger's solution, buffered saline, an albumin infusion solution, adextrose solution, a maltodextrin solution, glycerol, and ethanol, andthese materials may be used alone or in any combination thereof. Ifnecessary, other common additives, such as antioxidants, buffers,bacteriostatic agents, etc., may be added. Further, diluents,dispersants, surfactants, binders, and lubricants may be additionallyadded to prepare injectable formulations such as aqueous solutions,suspensions, and emulsions, or pills, capsules, granules, or tablets.Furthermore, the composition may be preferably formulated, dependingupon diseases and ingredients, using any appropriate method known in theart, as disclosed in Remington's Pharmaceutical Science, Mack PublishingCompany, Easton Pa.

Preferably, the treatment or treating mean improving or stabilizing thesubject's condition or disease; or preventing or relieving thedevelopment or worsening of symptoms associated with the subject'scondition or disease. The prevention, prophylaxis and preventivetreatment are used herein as synonyms.

Preferably, the treating or preventing of cancers or diseases associatedwith an angiogenic disorder may be any one or more of the following:alleviating one or more symptoms of angiogenesis, delaying progressingof angiogenesis, shrinking tumor size in patient having angiogenicdisorder, inhibiting tumor growth, prolonging overall survival,prolonging disease-free survival, prolonging time to diseaseprogression, preventing or delaying tumor metastasis, reducing oreradiating preexisting tumor metastasis, reducing incidence or burden ofpreexisting tumor metastasis, preventing recurrence of tumor.

The subject and patient are used herein interchangeably. They refer to ahuman or another mammal (e.g., mouse, rat, rabbit, dog, cat, cattle,swine, sheep, horse or primate) that can be afflicted with or issusceptible to a disease or disorder but may or may not have the diseaseor disorder. In certain embodiments, the subject is a human being.

Preferably, the amount effective or effective amount is the amount of anactive ingredient or a pharmaceutical composition disclosed herein thatwhen administered to a subject for treating a disease, is sufficient toeffect such treatment of the disease. Any improvement in the patient isconsidered sufficient to achieve treatment. An effective amount of anactive ingredient or a pharmaceutical composition disclosed herein, usedfor the treating or preventing of cancers or diseases associated with anangiogenic disorder may vary depending upon the manner ofadministration, the age, body weight, and general health of the patient.Ultimately, the prescribers or researchers will decide the appropriateamount and dosage regimen.

In the treatment or prevention method according to one embodimentdisclosed in the present application, the composition including theiCP-SOCS3 recombinant protein as an active ingredient may beadministered in a common manner via oral, buccal, rectal, intravenous,intra-arterial, intraperitoneal, intramuscular, intrasternal,percutaneous, topical, intraocular or subcutaneous route, morepreferably via intraperitoneal, intravenous, or intra-arterial injectionroute.

Advantageous Effects

According to one aspect disclosed in the present application,development and establishment of improved cell-permeable SOCS3recombinant protein, as therapeutics of cancers or diseases associatedwith an angiogenic disorder are provided. Because iCP-SOCS3 was designedbased on endogenous proteins, it would be a safety anti-angiogenic drugwithout side-effect.

However, the effects of the disclosures in the present application arenot limited to the above-mentioned effects, and another effects notmentioned will be clearly understood by those skilled in the art fromthe following description.

DESCRIPTION OF DRAWINGS

FIG. 1 shows Structure of aMTD- or rPeptide-Fused Recombinant Proteins.A schematic diagram of the His-tagged CRA recombinant proteins isillustrated and constructed according to the present invention. Thehis-tag for affinity purification (white), aMTD or rPeptide (gray) andcargo A (CRA, black) are shown.

FIG. 2a shows Construction of Expression Vectors for aMTDs- orrPeptide-Fused Recombinant Proteins. FIGS. 2b and 2c show the agarosegel electrophoresis analysis showing plasmid DNA fragments at 645 bpinsert encoding aMTDs or rPeptide-fused CRA cloned into the pET28a(+)vector according to the present invention.

FIGS. 3a to 3d show Inducible Expression of aMTD- or rPeptide-FusedRecombinant Proteins. Expressed recombinant aMTD- or randompeptide-fused CRA recombinant proteins were transformed in E. coli BL21(DE3) strain. Expression of recombinant proteins in E. coli before (−)and after (+) induction with IPTG was monitored by SDS-PAGE, and stainedwith Coomassie blue.

FIGS. 4a and 4b show Purification of aMTD- or rPeptide-Fused RecombinantProteins. Expressed recombinant proteins were purified by Ni²+ affinitychromatography under the natural condition. Purification of recombinantproteins displayed through SDS-PAGE analysis.

FIGS. 5a to 5u show Determination of aMTD-Mediated Cell-Permeability.Cell-permeability of a negative control (A: rP38) and referencehydrophobic CPPs (MTM12 and MTD85) are shown. The cell-permeability ofeach aMTD and/or rPeptide is visually compared to that of the cargoprotein lacking peptide sequence (HCA). Gray shaded area representsuntreated RAW 264.7 cells (vehicle); thin light gray line represents thecells treated with equal molar concentration of FITC (FITC only); darkthick line indicates the cells treated with FITC-his-tagged CRA protein(HCA); and the cells treated with the FITC-proteins (HMCA) fused tonegative control (rP38), reference CPP (MTM12 or MTD85) or newhydrophobic CPP (aMTD) are shown with light thick line and indicated byarrows.

FIGS. 6a to 6c show Determination of rPeptide-MediatedCell-Permeability. The cell-permeability of each aMTD and/or rPeptidewas visually compared to that of the cargo protein lacking peptidesequence (HCA). Gray shaded area represents untreated RAW 264.7 cells(vehicle); thin light gray line represents the cells treated with equalmolar concentration of FITC (FITC only); dark thick line indicates thecells treated with FITC-his-tagged CRA protein (HCA); and the cellstreated with the FITC-proteins fused to rPeptides are shown with lightthick line and indicated by arrows.

FIGS. 7a to 7k show Visualized Cell-Permeability of aMTD-FusedRecombinant Proteins. NIH3T3 ells were treated with FITC-labeled protein(10 μM) fused to aMTD for 1 hour at 37. Cell-permeability of theproteins was visualized by laser scanning confocal microscopy (LSM700version).

FIG. 8 shows Visualized Cell-Permeability of rPeptide-Fused RecombinantProteins. Cell-permeability of rPeptide-fused recombinant proteins wasvisualized by laser scanning confocal microscopy (LSM700 version).

FIGS. 9a to 9c show Relative Cell-Permeability of aMTD-Fused RecombinantProteins Compared to Negative Control (rP38). The FIG shows graphscomparing the cell-permeability of the recombinant proteins fused toaMTDs and a negative control (A: rP38).

FIGS. 10a to 10c show Relative Cell-Permeability of aMTD-FusedRecombinant Proteins Compared to Reference CPP (MTM12). The FIG showsgraphs comparing the cell-permeability of the recombinant proteins fusedto aMTDs and a reference CPP (MTM12).

FIGS. 11a to 11c show Relative Cell-Permeability of aMTD-FusedRecombinant Proteins Compared to Reference CPP (MTD85). The FIG showsgraphs comparing the cell-permeability of the recombinant proteins fusedto aMTDs and a reference CPP (MTD85).

FIG. 12 shows Relative Cell-Permeability of rPeptide-MediatedRecombinant Proteins Compared to Average that of aMTDs. The FIG showsgraphs comparing the cell-permeability of the recombinant proteins fusedto rPeptides and that (average value: aMTD AVE) of aMTDs.

FIGS. 13a to 13d show Association of Cell-Permeability with Amino AcidComposition in aMTD Sequences. These graphs display delivery potential(Geometric Mean) of aMTDs influenced with amino acid composition (A, I,V and L).

FIGS. 14a to 14d show Association of Cell-Permeability with CriticalFactors in aMTDs. These graphs show the association of cell-permeabilitywith critical factors [bending potential: proline position (PP),rigidity/flexibility: instability index (II), structural feature:aliphatic index (AI) and hydropathy: grand average of hydropathy(GRAVY)].

FIGS. 15a to 15d show Relative Relevance of aMTD-MediatedCell-Permeability with Critical Factors. Cell-permeability of 10 highand 10 low ranked aMTDs in their delivery potential were examined fortheir association with the critical factors [bending potential: prolineposition (PP), rigidity/flexibility: instability index (II), structuralfeature: aliphatic index (AI) and hydropathy: grand average ofhydropathy (GRAVY)].

FIG. 16 shows Relative Relevance of rPeptide-Mediated Cell-Permeabilitywith Hydropathy Range (GRAVY). This graph and a chart illustraterelative relevance of rPeptide-mediated cell-permeability with itshydropathy range (GRAVY).

FIG. 17 shows a structure of iCP-SOCS3 recombinant protein designedaccording to example 6-1.

FIG. 18 shows the agarose gel electrophoresis analysis showing plasmidDNA fragments insert encoding His-SOCS3-SDB (HS3B),His-aMTD165-SOCS3-SDB (HM₁₆₅S3B), His-aMTD165-SOCS3-SDC (HM₁₆₅S3C),His-aMTD₁₆₅-SOCS3-SDD (HM₁₆₅S3D), His-aMTD₁₆₅-SOCS3-SDE (HM₁₆₅S3E)cloned into the pET28a (+) vector according to example 6-1.

FIG. 19 shows inducible expression and purification of iCP-SOCS3recombinant protein in E. coli according to example 6-2 and improvementof solubility/yield of iCP-SOCS3 recombinant protein by fusing aMTD/SDaccording to example 6-3.

FIG. 20 shows aMTD-Mediated cell-permeability of SOCS3 recombinantproteins in RAW 264.7 cells according to example 7-1

FIG. 21 shows aMTD-Mediated intracellular delivery and localization ofSOCS3 Recombinant Proteins in NIH3T3 cells according to example 7-1.

FIG. 22 shows systemic delivery of aMTD/SD-fused SOCS3 recombinantproteins in vivo according to example 7-2.

FIG. 23 shows inhibition of IFN-γ-induced STAT phosphorylation byiCP-SOCS3 recombinant protein according to example 8-1.

FIG. 24 shows inhibition of LPS-induced cytokines secretion by iCP-SOCS3recombinant protein according to example 8-2.

FIG. 25 shows the structures of SOCS3 recombinant protein lacking aMTDprepared as a negative control according to example 9.

FIG. 26 shows expression, purification, and solubility/yield of HS3(lacking aMTD and SD) and HS3B (lacking aMTD) determined according toexample 6-3.

FIG. 27 shows the agarose gel electrophoresis analysis showing plasmidDNA fragments insert encoding His-aMTD #-SOCS3-SDB (HM # S3B) and His-rP#-SOCS3-SDB cloned into the pET28a (+) vector according to example 10.

FIGS. 28a and 28b show expression, purification, and solubility/yield ofHis-aMTD #-SOCS3-SDB (HM # S3B) determined according to example 10.

FIG. 29 shows expression, purification, and solubility/yield of His-rP#-SOCS3-SDB (HrP # S3B) determined according to example 10.

FIG. 30 shows solubility/yield of His-aMTD #-SOCS3-SDB (HM # S3B)determined according to example 10.

FIG. 31 show aMTD-mediated cell-permeability. The cell-permeability ofeach SOCS3 recombinant protein fused with SD and various aMTD isvisually compared to that of the cargo protein lacking CPP (HS3B) orlacking CPP and SD (HS3). Gray shaded area represents untreated E. colicells (diluent); line 1 represents the cells treated with equal molarconcentration of FITC (FITC only); line 2 indicates the cells treatedwith FITC-his-SOCS protein (FITC-HS3); line 3 indicates the cellstreated with FITC-his-SOCS-SDB protein (FITC-HS3B) line 4 indicates thecells treated with FITC-his-aMTD #-SOCS-SDB protein (FITC-HM # S3B).

FIG. 32 shows relative cell-permeability of His-aMTD #-SOCS3-SDB-Fusedrecombinant proteins Compared to control (Vehicle, FITC only, HS3 andHS3B).

FIG. 33 shows random Peptide-Mediated cell-permeability. Thecell-permeability of each SOCS3 recombinant protein fused with SDB andaMTD₁₆₅ or various rP is visually compared to that of the cargo proteinlacking CPP (HS3B) or lacking CPP and SD (HS3). Gray shaded arearepresents untreated E. coli cells (diluent); line 1 represents thecells treated with equal molar concentration of FITC (FITC only); line 2indicates the cells treated with FITC-his-SOCS protein (FITC-HS3); line3 indicates the cells treated with FITC-his-SOCS-SDB protein (FITC-HS3B)and line 4 indicates the cells treated withFITC-his-rPeptide_(#)-SOCS-SDB protein (FITC-HrP_(#) S3B).

FIG. 34 shows relative cell-permeability of His-rP_(#)-SOCS3-SDB-Fusedrecombinant proteins Compared to control (Vehicle, FITC only, HS3 andHS3B).

FIG. 35 shows apoptotic cells analysis according to example 11-1.

FIG. 36 shows induction of apoptosis by iCP-SOCS3 recombinant proteinsaccording to example 11-2.

FIG. 37 shows cell migration inhibition by iCP-SOCS3 recombinant proteinaccording to example 11-3.

FIG. 38 shows solubility/yield, permeability and biological activity ofHis-aMTD #-SOCS3-SDB (HM # S3B) determined according to example 10 to11-3.

FIG. 39a shows a structure of M₁₆₅S3SB (lacking his-tag) determinedaccording to example 12-1; and FIG. 39b shows expression, purification,and solubility/yield of M₁₆₅S3SB (lacking his-tag) determined accordingto example 12-1.

FIG. 40 shows cell-permeability of SOCS3 recombinant proteins (lackinghis-tag) in RAW 264.7 cells according to example 12-2.

FIG. 41 shows Annexin V analysis according to example 12-3.

FIG. 42 shows cell migration inhibition (bottom) by iCP-SOCS3recombinant protein according to example 12-3.

FIG. 43 shows a structure of iCP-SOCS3 recombinant protein(His-aMTD₁₆₅-SOCS3-SDB) constructed according to example 12-4.

FIG. 44 shows expression, purification, and solubility/yield ofHM₁₆₅S3SB and HM₁₆₅S3SB′ determined according to example 12-4.

FIG. 45 shows aMTD-Mediated cell-permeability of iCP-SOCS3 recombinantproteins (HM₁₆₅S3B and HM₁₆₅S3B′(V28L)) in RAW 264.7 cells according toexample 12-5.

FIG. 46 shows antiproliferative activity of iCP-SOCS3 recombinantproteins (HM₁₆₅S3B and HM₁₆₅S3B′(V28L)) according to example 12-6.

FIG. 47 shows induction of apoptosis by iCP-SOCS3 recombinant proteins(HM₁₆₅S3B and HM₁₆₅S3B′(V28L)) according to example 12-6.

FIGS. 48a and 48b show cell migration inhibition by SOCS3 recombinantproteins (HM₁₆₅S3B and HM₁₆₅S3B′(V28L)) according to example 12-6.

FIG. 49a shows a structure of iCP-SOCS3 recombinant proteins to example12-7. FIG. 49b shows agarose gel electrophoresis analysis according toexample 12-7[[.]] FIG. 49c shows inducible expressions and purificationsof iCP-SOCS3 recombinant protein in E. coli (bottom) according toexample 12-7.

FIG. 50 shows inhibition of IFN-γ-induced STAT phosphorylation byiCP-SOCS3 recombinant protein according to example 13.

FIGS. 51a and 51b show an effect of treating EDTA and proteinase K,respectively, on aMTD-mediated SOCS3 protein uptake into cells accordingto example 14-1.

FIGS. 52a and 52b show an effect of treating taxol and antimycin onaMTD-mediated SOCS3 protein uptake into cells according to example 14-1.

FIG. 53 shows an effect of temperature on aMTD-mediated SOCS3 proteinuptake into cells according to example 14-1.

FIG. 54 shows aMTD-mediated cell-to-cell delivery assessed according toexample 14-1.

FIG. 55 shows bioavailability of iCP-SOCS3 recombinant protein in PBMC,splenocytes and hepatocytes analyzed by fluorescence microscopyaccording to example 15.

FIG. 56 shows biodistribution of iCP-SOCS3 recombinant protein inpancreas tissues analyzed by confocal microscope according to example16.

FIG. 57 shows anti-proliferative activity of iCP-SOCS3 recombinantprotein in endothelial cells according to example 17-1.

FIG. 58 shows cell migration inhibition activity by iCP-SOCS3recombinant protein in endothelial cells according to example 17-2.

FIG. 59 shows decrease in branch points and tube length by iCP-SOCS3recombinant protein in endothelial cells analyzed according to example17-3.

FIG. 60 shows down regulation of angiogenesis-associated proteins byiCP-SOCS3 recombinant proteins in growth factor-stimulated endothelialcells determined by immunoblotting according to example 17-4.

FIG. 61 shows suppression of neovasculization by iCP-SOCS3 recombinantproteins in eggs assessed according to example 17-6.

FIG. 62 shows suppression of cancer cell-mediated invasion by iCP-SOCS3recombinant proteins in endothelial cells assessed according to example18-1.

FIG. 63 shows suppression of spheroid size by iCP-SOCS3 recombinantproteins according to example 18-2.

FIG. 64 shows suppression of sprouts number and length by iCP-SOCS3recombinant proteins according to example 18-2.

FIG. 65 shows suppression of tumor-mediated angiogenesis by iCP-SOCS3recombinant proteins according to example 18-3.

FIGS. 66 and 67 show suppression of the tumor growth andangiogenesis-associated proteins by iCP-SOCS3 recombinant proteins inhepatocellular carcinoma xenograft models according to example 19.

FIG. 68 shows suppression of the tumor growth andangiogenesis-associated proteins by iCP-SOCS3 recombinant proteins inpancreatic cancer xenograft models according to example 19.

FIG. 69 shows suppression of the tumor growth andangiogenesis-associated proteins by iCP-SOCS3 recombinant proteins inglioblastoma xenograft according to example 19.

FIG. 70 shows humanized SDB domain according to example 12-4.

FIG. 71 shows sequences of amino acid and nucleotide of basic CPP, andprimers used in example 6-4.

FIG. 72a shows a structure of aMTD/SD-fused SOCS3 recombinant proteinand basic CPP/SD-fused SOCS3 recombinant protein according to example6-4; and FIG. 72b shows expression, purification and solubility/yield ofaMTD/SD-fused SOCS3 recombinant protein and basic CPP/SD-fused SOCS3recombinant protein analyzed according to example 6-4.

FIGS. 73a and 73b show comparison of cell-permeability between aMTD/SDfused SOCS3 recombinant proteins and basic CPP/SD-fused in RAW 264.7cells according to example 7-1-2.

FIG. 74 shows comparison of tissue-permeability between aMTD/SD fusedSOCS3 recombinant proteins and basic CPP/SD-fused in various tissues ofICR mice according to example 7-2-2.

FIGS. 75a and 75b show an effect of treating proteinase K (A) and Taxol(B) on aMTD (or basic CPP)-mediated SOCS3 protein uptake into cellsaccording to example 14-2.

FIGS. 76 and 77 show aMTD (or basic CPP)-mediated cell-to-cell delivery(FIG. 76) and cell-to-cell function (FIG. 77) assessed according toexample 14-2.

FIGS. 78a and 78b show dose-dependency of cell-permeability of iCP-SOCS3recombinant proteins analyzed according to example 14-3.

FIGS. 79a and 79b show time-dependency of cell-permeability of theiCP-SOCS3 recombinant proteins analyzed according to example 14-3.

FIG. 80 shows angiogenesis-inhibitory efficacy of iCP-SOCS3 recombinantproteins analyzed according to example 17-5.

FIG. 81 shows reduction of the expression levels of angiogenesis-relatedfactors in the iCP-SOCS3 recombinant protein-treated groups according toexample 18-3.

FIGS. 82 and 83 illustrate developments of iCP-SOCS3 recombinantprotein.

FIG. 84 illustrates the selection of 5 kinds of random peptides that donot satisfying one or more critical factors.

MODE FOR INVENTION

1. Analysis of Reference Hydrophobic CPPs to Identify ‘Critical Factors’for Development of Advanced MTDs

Previously reported MTDs were selected from a screen of more than 1,500signal peptide sequences. Although the MTDs that have been developed didnot have a common sequence or sequence motif, they were all derived fromthe hydrophobic (H) regions of signal sequences (HRSSs) that also lackcommon sequences or motifs except their hydrophobicity and the tendencyto adopt alpha-helical conformations. The wide variation in H-regionsequences may reflect prior evolution for proteins with membranetranslocating activity and subsequent adaptation to the SRP/Sec61machinery, which utilizes a methionine-rich signal peptide bindingpocket in SRP to accommodate a wide-variety of signal peptide sequences.

Previously described hydrophobic CPPs (e.g. MTS/MTM and MTD) werederived from the hydrophobic regions present in the signal peptides ofsecreted and cell surface proteins. The prior art consists first, of adhoc use of H-region sequences (MTS/MTM), and second, of H-regionsequences (with and without modification) with highest CPP activityselected from a screen of 1,500 signal sequences (MTM). Second priorart, the modified H-region derived hydrophobic CPP sequences hadadvanced in diversity with multiple number of available sequences apartfrom MTS/MTM derived from fibroblast growth factor (FGF) 4. However, thenumber of MTDs that could be modified from naturally occurring secretedproteins are somewhat limited. Because there is no set of rules indetermining their cell-permeability, no prediction for thecell-permeability of modified MTD sequences can be made before testingthem.

The hydrophobic CPPs, like the signal peptides from which theyoriginated, did not conform to a consensus sequence, and they hadadverse effects on protein solubility when incorporated into proteincargo. We therefore set out to identify optimal sequence and structuraldeterminants, namely critical factors (CFs), to design new hydrophobicCPPs with enhanced ability to deliver macromolecule cargoes includingproteins into the cells and tissues while maintaining proteinsolubility. These newly developed CPPs, advanced macromoleculetransduction domains (aMTDs) allowed almost infinite number of possibledesigns that could be designed and developed based on the criticalfactors. Also, their cell-permeability could be predicted by theircharacter analysis before conducting any in vitro and/or in vivoexperiments. These critical factors below have been developed byanalyzing all published reference hydrophobic CPPs.

1-1. Analysis of Hydrophobic CPPs

Seventeen different hydrophobic CPPs (Table 1) published from 1995 to2014 (Table 2) were selected. After physiological and chemicalproperties of selected hydrophobic CPPs were analyzed, 11 differentcharacteristics that may be associated with cell-permeability have beenchosen for further analysis. These 11 characteristics are as follows:sequence, amino acid length, molecular weight, pI value, bendingpotential, rigidity/flexibility, structural feature, hydropathy, residuestructure, amino acid composition and secondary structure of thesequences (Table 3-land Table 3-2).

Table 1 shows the summary of published hydrophobic Cell-PenetratingPeptides which were chosen.

TABLE 1 # Pepides Origin Protein Ref. 1 MTM Homo sapiens NP_001998Kaposi fibroblast growth factor (K-FGF) 1 2 MTS Homo sapiens NP_001998Kaposi fibroblast growth factor (K-FGF) 2 3 MTD10 Streptomycescoelicolor NP_625021 Glycosyl hydrolase 8 4 MTD13 Streptomycescoelicolor NP_639877 Putative secreted protein 3 5 MTD47 Streptomycescoelicolor NP_627512 Secreted protein 4 6 MTD56 Homo sapiens P23274Peptidyl-prolyl cis-trans isomerase B precursor 5 7 MTD73 Drosophilamelanogaster AAA17887 Spatzle (spz) protein 5 8 MTD77 Homo sapiensNP_003231 Kaposi fibroblast growth factor (K-FGF) 6 9 MTD84 Phytophthoracactorum AAK63068 Phytotoxic protein PcF precusor 4 10 MTD85Streptomyces coelicolor NP_629842 Peptide transport system peptidebinding 7 protein 11 MTD86 Streptomyces coelicolor NP_629842 Peptidetransport system secreted peptide 7 binding protein 12 MTD103 Homosapiens TMBV19 domain Family member B 8 13 MTD132 Streptomycescoelicolor NP_628377 P60-family secreted protein 4 14 MTD151Streptomyces coelicolor NP_630126 Secreted chitinase 8 15 MTD173Streptomyces coelicolor NP_624384 Secreted protein 4 16 MTD174Streptomyces coelicolor NP_733505 Large, multifunctional secretedprotein 8 17 MTD181 Neisseria meningitidis Z2491 CAB84257.1 Putativesecreted protein 4

Table 2 summarizes reference information.

TABLE 2 References # Title Journal Year Vol Issue Page 1 Inhibition ofNuclear Translocation of JOURNAL OF 1995 270 24 14255 TranscriptionFactor NF-kB by a Synthetic BIOLOGICAL peptide Containing a CellCHEMISTRY Membrane-permeable Motif and Nuclear Localization Sequence 2Epigenetic Regulation of Gene Structure and NATURE 2001 19 10 929Function with a Cell-Permeable Cre BIOTECHNOLOGY Recombinase 3Cell-Permeable NM23 Blocks the Maintenance CANCER 2011 71 23 7216 andProgression of Established Pulmonary RESEARCH Metastasis 4 AntitumorActivity of Cell-Permeable MOLECULAR 2012 20 8 1540 p18INK4c WithEnhanced Membrane and THERAPY Tissue Penetration 5 Antitumor Activity ofCell-Permeable RUNX3 CLINICAL 2012 19 3 680 Protein in Gastric CancerCells CANCER RESEARCH 6 The Effect of Intracellular Protein Delivery onBIOMATERIALS 2013 34 26 6261 the Anti-Tumor Activity of RecombinantHuman Endostatin 7 Partial Somatic to Stem Cell TransformationsSCIENTIFIC 2014 4 10 4361 Induced By Cell-Permeable ReprogrammingREPORTS Factors 8 Cell-Permeable Parkin Proteins Suppress PLOS ONE 20149 7 17 Parkinson Disease-Associated Phenotypes in Cultured Cells andAnimals

Table 3 shows characteristics of published hydrophobic Cell-PenetratingPeptides (A) which were analyzed.

TABLE 3 Rigidity/ Structural Flexibility Feature SEQ (Instability(Aliphatic ID Molecular Bending Index: Index: Hydropathy # NO PeptidesSequence Length Weight pI Potential II) AI) (GRAVY) 1 839 MTMAAVALLPAVLL 16 1,515.9 5.6 Bending 45.5 220.0 2.4 ALLAP 2 840 MTSAAVLLPVLLAAP 12 1,147.4 5.6 Bending 57.3 211.7 2.3 3 841 MTD10LGGAVVAAPV 16 1,333.5 5.5 Bending 47.9 140.6 1.8 AAAVAP 4 842 MTD13LAAAALAVLPL 11 1,022.3 5.5 Bending 26.6 213.6 2.4 5 843 MTD47 AAAVPVLVAA10 881.0 5.6 Bending 47.5 176.0 2.4 6 844 MTD56 VLLAAALIA 9 854.1 5.5No-Bending 8.9 250.0 3.0 7 845 MTD73 PVLLLLA 7 737.9 6.0 No-Bending 36.1278.6 2.8 8 846 MTD77 AVALLILAV 9 882.1 5.6 No-Bending 30.3 271.1 3.3 9847 MTD84 AVALVAVVAVA 11 982.2 5.6 No-Bending 9.1 212.7 3.1 10 848 MTD85LLAAAAALLLA 11 1,010.2 5.5 No-Bending 9.1 231.8 2.7 11 849 MTD86LLAAAAALLLA 11 1,010.2 5.5 No-Bending 9.1 231.8 2.7 12 850 MTD103LALPVLLLA 9 922.2 5.5 Bending 51.7 271.1 2.8 13 851 MTD132 AVVVPAIVLAAP12 1,119.4 5.6 Bending 50.3 195.0 2.4 14 852 MTD151 AAAPVAAVP 9 1,031.45.5 Bending 73.1 120.0 1.6 15 853 MTD173 AVIPILAVP 9 892.1 5.6 Bending48.5 216.7 2.4 16 854 MTD174 LILLLPAVALP 12 1,011.8 5.5 Bending 79.1257.3 2.6 17 855 MTD181 AVLLLPAAA 9 838.0 5.6 Bending 51.7 206.7 2.4 AVE10.8 ± 1,011 ± 5.6 ± 0.1 Proline 40.1 ± 21.9 217.9 ± 43.6 2.5 ± 0.4 2.4189.6 Presence Residue A/a Composition Secondary # Peptides Structure AV L I P G Structure Cargo Ref. 1 MTM Aliphatic Ring 6 2 6 0 2 0 Helixp50 1 2 MTS ″ 4 2 4 0 2 0 No-Helix CRE 2 3 MTD10 ″ 7 4 1 0 2 2 HelixParkin 8 4 MTD13 ″ 5 1 4 0 1 0 No-Helix RUNX3 3 5 MTD47 ″ 5 3 1 0 1 0No-Helix CMYC 4 6 MTD56 ″ 4 1 3 1 0 0 Helix ES 5 7 MTD73 ″ 1 1 4 0 1 0Helix ES 5 8 MTD77 ″ 3 2 3 1 0 0 Helix NM23 6 9 MTD84 ″ 5 5 1 0 0 0Helix OCT4 4 10 MTD85 ″ 6 0 5 0 0 0 No-Helix RUNX3 7 11 MTD86 ″ 6 0 5 00 0 No-Helix SOX2 7 12 MTD103 ″ 2 1 5 0 1 0 Helix p18 8 13 MTD132 ″ 4 41 1 2 0 No-Helix LIN28 4 14 MTD151 ″ No-Helix Parkin 8 15 MTD173 ″ 2 2 12 2 0 Helix KLF4 4 16 MTD174 ″ Helix Parkin 8 17 MTD181 ″ 4 1 3 0 1 0No-Helix SOX2 4

Two peptide/protein analysis programs were used (ExPasy: SoSui:harrier.nagahama-i-bio.acjp) to determine various indexes and structuralfeatures of the peptide sequences and to design new sequence. Followingsare important factors analyzed.

1-2. Characteristics of Analyzed Peptides: Length, Molecular Weight andPl Value

Average length, molecular weight and pl value of the peptides analyzedwere 10.8±2.4, 1,011±189.6 and 5.6±0.1, respectively (Table 4)

Table 4 summarizes Critical Factors (CFs) of published hydrophobicCell-Penetrating Peptides (A) which were analyzed.

TABLE 4 Length: 10.8 ± 2.4 Molecular Weight: 1,011 ± 189.6 pl: 5.6 ± 0.1Bending Potential (BP): Proline presences in the middle and/or the endof peptides, or No Proline Instability Index (II): 40.1 ± 21.9 ResidueStructure & Aliphatic Index (AI): 217.9 ± 43.6 Hydropathy (GRAVY): 2.5 ±0.4 Aliphatic Ring: Non-polar hydrophobic & aliphatic amino acid (A, V,L, I) Secondary Structure: α-Helix is favored but not required

1-3. Characteristics of Analyzed Peptides: Bending Potential—ProlinePosition (PP)

Bending potential (bending or no-bending) was determined based on thefact whether proline (P) exists and/or where the amino acid(s) providingbending potential to the peptide in recombinant protein is/are located.Proline differs from the other common amino acids in that its side chainis bonded to the backbone nitrogen atom as well as the alpha-carbonatom. The resulting cyclic structure markedly influences proteinarchitecture which is often found in the bends of folded peptide/proteinchain.

Eleven out of 17 were determined as ‘Bending’ peptide which means thatproline is present in the middle of sequence for peptide bending and/orlocated at the end of the peptide for protein bending. As indicatedabove, peptide sequences could penetrate the plasma membrane in a “bent”configuration. Therefore, bending or no-bending potential is consideredas one of the critical factors for the improvement of currenthydrophobic CPPs.

1-4. Characteristics of Analyzed Peptides:Rigidity/Flexibility—Instability Index (II)

Since one of the crucial structural features of any peptide is based onthe fact whether the motif is rigid or flexible, which is an intactphysicochemical characteristic of the peptide sequence, instabilityindex (II) of the sequence was determined. The index value representingrigidity/flexibility of the peptide was extremely varied (8.9-79.1), butaverage value was 40.1±21.9 which suggested that the peptide should besomehow flexible, but not too much rigid or flexible (Table 3).

1-5. Characteristics of Analyzed Peptides: StructuralFeatures—Structural Feature (Aliphatic Index: AI) and Hydropathy (GrandAverage of Hydropathy: GRAVY)

Alanine (V), valine (V), leucine (L) and isoleucine (I) containaliphatic side chain and are hydrophobic—that is, they have an aversionto water and like to cluster. These amino acids having hydrophobicityand aliphatic residue enable them to pack together to form compactstructure with few holes. Analyzed peptide sequence showed that allcomposing amino acids were hydrophobic (A, V, L and I) except glycine(G) in only one out of 17 (MTD10-Table 3) and aliphatic (A, V, L, I, andP). Their hydropathic index (Grand Average of Hydropathy: GRAVY) andaliphatic index (AI) were 2.5±0.4 and 217.9±43.6, respectively. Theiramino acid composition is also indicated in the Table 3.

1-6. Characteristics of Analyzed Peptides: Secondary Structure(Helicity)

As explained above, the CPP sequences may be supposed to penetrate theplasma membrane directly after inserting into the membranes in a “bent”configuration with hydrophobic sequences having α-helical conformation.In addition, our analysis strongly indicated that bending potential wascrucial for membrane penetration. Therefore, structural analysis of thepeptides was conducted to determine whether the sequences were to formhelix or not. Nine peptides were helix and eight were not (Table 3). Itseems to suggest that helix structure may not be required.

1-7. Determination of Critical Factors (CFs)

In the 11 characteristics analyzed, the following 6 are selected namely“Critical Factors” for the development of new hydrophobic CPPs—advancedMTDs: amino acid length, bending potential (proline presence andlocation), rigidity/flexibility (instability index: II), structuralfeature (aliphatic index: AI), hydropathy (GRAVY) and amino acidcomposition/residue structure (hydrophobic and aliphatic A/a) (Table 3and Table 4).

2. Analysis of Selected Hydrophobic CPPs to Optimize ‘Critical Factors’

Since the analyzed data of the 17 different hydrophobic CPPs (analysisA, Table 3 and 4) previously developed during the past 2 decades showedhigh variation and were hard to make common- or consensus—features,analysis B (Table 5 and 6) and C (Table 7 and 8) were also conducted tooptimize the critical factors for better design of improved CPPs—aMTDs.Therefore, 17 hydrophobic CPPs have been grouped into two groups andanalyzed the groups for their characteristics in relation to the cellpermeable property. The critical factors have been optimized bycomparing and contrasting the analytical data of the groups anddetermining the common homologous features that may be critical for thecell permeable property.

2-1. Selective Analysis (B) of Peptides Used to Biologically ActiveCargo Protein for In Vivo

In analysis B, eight CPPs were used with each biologically active cargoin vivo. Length was 11±3.2, but 3 out of 8 CPPs possessed little bendingpotential. Rigidity/Flexibility (instability index: II) was 41±15, butremoving one [MTD85: rigid, with minimal II (9.1)] of the peptidesincreased the overall instability index to 45.6±9.3. This suggested thathigher flexibility (40 or higher II) is potentially be better. All othercharacteristics of the 8 CPPs were similar to the analysis A, includingstructural feature and hydropathy (Tables 5-1, 5-2, and 6)

Table 5 shows characteristics of published hydrophobic Cell-PenetratingPeptides (B): selected CPPs that were used to each cargo in vivo.

TABLE 5 Rigidity/ Structural Flexibility Feature Molecular Bending(Instability (Aliphatic Hydropathy # Peptides Sequence Length Weight pIPotential Index: II) Index: AI) (GRAVY) 1 MTM AAVALLPAVLLALLAP 161,515.9 5.6 Bending 45.5 220.0 2.4 2 MTS AAVLLPVLLAAP 12 1,147.4 5.6Bending 57.3 211.7 2.3 3 MTD10 LGGAVVAAPVAAAVAP 16 1,333.5 5.5 Bending47.9 140.6 1.8 4 MTD73 PVLLLLA 7 737.9 6.0 No- 36.1 278.6 2.8 Bending 5MTD77 AVALLILAV 9 882.1 5.6 No- 30.3 271.1 3.3 Bending 6 MTD85LLAAAAALLLA 11 1,010.2 5.5 No- 9.1* 231.8 2.7 Bending 7 MTD103 LALPVLLLA9 922.2 5.5 Bending 51.7 271.1 2.8 8 MTD132 AVVVPAIVLAAP 12 1,119.4 5.6Bending 50.3 195.0 2.4 AVE 11 ± 3.2 1,083 ± 252 5.6 ± 0.1 Proline 41 ±15 227 ± 47 2.5 ± 0.4 Presence A/a Residue Composition Secondary #Peptides Structure A V L I P G Structure Cargo Ref. 1 MTM Aliphatic 6 26 0 2 0 Helix p50 1 Ring 2 MTS ″ 4 2 4 0 2 0 No-Helix CRE 2 3 MTD10 ″ 74 1 0 2 2 Helix Parkin 8 4 MTD73 ″ 1 1 4 0 1 0 Helix ES 6 5 MTD77 ″ 3 23 1 0 0 Helix NM23 3 6 MTD85 ″ 6 0 5 0 0 0 No-Helix RUNX3 5 7 MTD103 ″ 21 5 0 1 0 Helix p18 4 8 MTD132 ″ 4 4 1 1 2 0 No-Helix LIN28 7 *Removingthe MTD85 increases II to 45.6 ± 9.3

Table 6 shows summarized Critical Factors of published hydrophobicCell-Penetrating Peptides (B).

TABLE 6 Length: 11 ± 3.2 Molecular Weight: 1,083 ± 252 pl: 5.6 ± 0.1Bending Potential (BP): Proline presences in the middle and/or the endof peptides, or No Proline Instability Index (II): 41.0 ± 15 (Removingthe MTD85 increases II to 45.6 ± 9.3) Residue Structure & AliphaticIndex (AI): 227 ± 47 Hydropathy (GRAVY): 2.5 ± 0.4 Aliphatic Ring:Non-polar hydrophobic & aliphatic amino acid (A, V, L, I) SecondaryStructure: α-Helix is favored but not required

2-2. Selective Analysis (C) of Peptides that Provided Bending Potentialand Higher Flexibility

To optimize the ‘Common Range and/or Consensus Feature of CriticalFactor’ for the practical design of aMTDs and the random peptides (rPsor rPeptides), which were to prove that the ‘Critical Factors’determined in the analysis A, B and C were correct to improve thecurrent problems of hydrophobic CPPs—protein aggregation, lowsolubility/yield, and poor cell-/tissue-permeability of the recombinantproteins fused to the MTS/MTM or MTD, and non-common sequence andnon-homologous structure of the peptides, empirically selected peptideswere analyzed for their structural features and physicochemical factorindexes.

Hydrophobic CPPs which did not have a bending potential, rigid or toomuch flexible sequences (too much low or too much high InstabilityIndex), or too low or too high hydrophobic CPPs were unselected, butsecondary structure was not considered because helix structure ofsequence was not required.

In analysis C, eight selected CPP sequences that could provide a bendingpotential and higher flexibility were finally analyzed (Tables 7-1, 7-2,and 8). Common amino acid length is 12 (11.6±3.0). Proline is presencein the middle of and/or the end of sequence. Rigidity/Flexibility (II)is 45.5-57.3 (Avg: 50.1±3.6). AI and GRAVY representing structuralfeature and hydrophobicity of the peptide are 204.7±37.5 and 2.4±0.3,respectively. All peptides are consisted with hydrophobic and aliphaticamino acids (A, V, L, I, and P). Therefore, analysis C was chosen as astandard for the new design of new hydrophobic CPPs—aMTDs.

Table 7 shows characteristics of published hydrophobic Cell-PenetratingPeptides (C): selected CPPs that provided bending potential and higherflexibility.

TABLE 7 Rigidity/ Structural Flexibility Feature Molecular Bending(Instability (Aliphatic Hydropathy # Peptides Sequence Length Weight pIPotential Index: II) Index: AI) (GRAVY) 1 MTM AAVALLPAVLLALLAP 16 1515.95.6 Bending 45.5 220.0 2.4 2 MTS AAVLLPVLLAAP 12 1147.4 5.6 Bending 57.3211.7 2.3 3 MTD10 LGGAVVAAPVAAAVAP 16 1333.5 5.5 Bending 47.9 140.6 1.84 MTD47 AAAVPVLVAA 10 881.0 5.6 Bending 47.5 176.0 2.4 5 MTD103LALPVLLLA 9 922.2 5.5 Bending 51.7 271.1 2.8 6 MTD132 AVVVPAIVLAAP 121119.4 5.6 Bending 50.3 195.0 2.4 7 MTD173 AVIPILAVP 9 892.1 5.6 Bending48.5 216.7 2.4 8 MTD181 AVLLLPAAA 9 838.0 5.6 Bending 51.7 206.7 2.4 AVE11.6 ± 1081.2 ± 5.6 ± 0.1 Proline 50.1 ± 3.6 204.7 ± 37.5 2.4 ± 0.3 3.0244.6 Presence A/a Residue Composition Secondary # Peptides Structure AV L I P G Structure Cargo Ref. 1 MTM Aliphatic 6 2 6 0 2 0 Helix p50 1Ring 2 MTS ″ 4 2 4 0 2 0 No-Helix CRE 2 3 MTD10 ″ 7 4 1 0 2 2 HelixParkin 8 4 MTD47 ″ 5 3 1 0 1 0 No-Helix CMYC 4 5 MTD103 ″ 2 1 5 0 1 0Helix p18 8 6 MTD132 ″ 4 4 1 1 2 0 No-Helix LIN28 4 7 MTD173 ″ 2 2 1 2 20 Helix KLF4 4 8 MTD181 ″ 4 1 3 0 1 0 No-Helix SOX2 4

Table 8 shows summarized Critical Factors of published hydrophobicCell-Penetrating Peptides (C).

TABLE 8 Length: 11.6 ± 3.0 Molecular Weight: 1,081.2 ± 224.6 pl: 5.6 ±0.1 Bending Potential (BP): Proline presences in the middle and/or theend of peptides, or No Proline Instability Index (II): 50.1 ± 3.6Residue Structure & Aliphatic Index (AI): 204.7 ± 37.5 Hydropathy(GRAVY): 2.4 ± 0.3 Aliphatic Ring: Non-polar hydrophobic & aliphaticamino acid (A, V, L, I) Secondary Structure: α-Helix is favored but notrequired

3. New Design of Improved Hydrophobic CPPs—aMTDs Based on the OptimizedCritical Factors

3-1. Determination of Common Sequence and/or Common Homologous Structure

As mentioned above, H-regions of signal sequence (HRSS)-derived CPPs(MTS/MTM and MTD) do not have a common sequence, sequence motif, and/orcommon-structural homologous feature. In this invention, the aim is todevelop improved hydrophobic CPPs formatted in the common sequence- andstructural-motif which satisfy newly determined ‘Critical Factors’ tohave ‘Common Function,’ namely, to facilitate protein translocationacross the membrane with similar mechanism to the analyzed referenceCPPs. Based on the analysis A, B and C, the common homologous featureshave been analyzed to determine the critical factors that influence thecell-permeability. The range value of each critical factor has beendetermined to include the analyzed index of each critical factor fromanalysis A, B and C to design novel aMTDs (Table 9). These features havebeen confirmed experimentally with newly designed aMTDs in theircell-permeability.

Table 9 shows comparison the range/feature of each Critical Factorbetween the value of analyzed CPPs and the value determined for newdesign of novel aMTDs sequences

TABLE 9 Summarized Critical Factors of aMTD Selected CPPs Newly DesignedCPPs Critical Factor Range Range Bending Potential Proline presencesProline presences (Proline Position: PP) in the middle in the middleand/or at the (5′, 6′, 7′ end of peptides or 8′) and at the end ofpeptides Rigidity/Flexibility 45.5-57.3 40-60 (Instability Index: II)(50.1 ± 3.6) Structural Feature 140.6-220.0 180-220 (Aliphatic Index:AI) (204.7 ± 37.5) Hydropathy 1.8-2.8 2.1-2.6 (Grand Average of  (2.4 ±0.3) Hydropathy GRAVY) Length 11.6 ± 3.0  9-13 (Number of Amino Acid)Amino acid Composition A, V, I, L, P A, V, I, L, P

In Table 9, universal common features and sequence/structural motif areprovided. Length is 9-13 amino acids, and bending potential is providedwith the presence of proline in the middle of sequence (at 5′, 6′, 7′ or8′ amino acid) for peptide bending and at the end of peptide forrecombinant protein bending and Rigidity/Flexibility of aMTDs is II>40are described in Table 9.

3-2. Critical Factors for Development of Advanced MTDs

Recombinant cell-permeable proteins fused to the hydrophobic CPPs todeliver therapeutically active cargo molecules including proteins intolive cells had previously been reported, but the fusion proteinsexpressed in bacteria system were hard to be purified as a soluble formdue to their low solubility and yield. To address the crucial weaknessfor further clinical development of the cell-permeable proteins asprotein-based biotherapeutics, greatly improved form of the hydrophobicCPP, named as advanced MTD (aMTD) has newly been developed throughcritical factors-based peptide analysis. The critical factors used forthe current invention of the aMTDs are herein (Table 9).

1. Amino Acid Length: 9-13

2. Bending Potential (Proline Position: PP)

: Proline presences in the middle (from 5′ to 8′ amino acid) and at theend of sequence

3. Rigidity/Flexibility (Instability Index: II): 40-60

4. Structural Feature (Aliphatic Index: AI): 180-220

5. Hydropathy (GRAVY): 2.1-2.6

6. Amino Acid Composition: Hydrophobic and Aliphatic amino acids—A, V,L, I and P

3-3. Design of Potentially Best aMTDs that all Critical Factors areConsidered and Satisfied

After careful consideration of six critical factors derived fromanalysis of unique features of hydrophobic CPPs, advanced macromoleculetransduction domains (aMTDs) have been designed and developed based onthe common 12 amino acid platform which satisfies the critical factorsincluding amino acid length (9-13) determined from the analysis.

Unlike previously published hydrophobic CPPs that require numerousexperiments to determine their cell-permeability, newly developed aMTDsequences could be designed by performing just few steps as followsusing above mentioned platform to follow the determined rangevalue/feature of each critical factor.

First, prepare the 12 amino acid sequence platform for aMTD. Second,place proline (P) in the end (12′) of sequence and determine where toplace proline in one of four U(s) in 5′, 6′, 7′, and 8. Third, alanine(A), valine (V), leucine (L) or isoleucine (I) is placed in either X(s)and/or U(s), where proline is not placed. Lastly, determine whether theamino acid sequences designed based on the platform, satisfy the valueor feature of six critical factors to assure the cell permeable propertyof aMTD sequences. Through these processes, numerous novel aMTDsequences have been constructed. The expression vectors for preparingnon-functional cargo recombinant proteins fused to each aMTD, expressionvectors have been constructed and forcedly expressed in bacterial cells.These aMTD-fused recombinant proteins have been purified in soluble formand determined their cell-permeability quantitatively. aMTD sequenceshave been newly designed, numbered from 1 to 240, as shown in Table10-15. In Table 10-15, sequence ID Number is a sequence listings forreference, and aMTD numbers refer to amino acid listing numbers thatactually have been used at the experiments. For further experiments,aMTD numbers have been used. In addition, polynucleotide sequences shownin the sequence lists have been numbered from SEQ ID NO: 241 to SEQ IDNO: 480.

Tables 10 to 15 shows 240 new hydrophobic aMTD sequences that weredeveloped to satisfy all critical factors.

TABLE 10 Rigidity/ Sturctural Sequence Flexibility Feature HydropathyResidue ID Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 1 1AAALAPVVLALP 12 57.3 187.5 2.1 Aliphatic 2 2 AAAVPLLAVVVP 12 41.3 195.02.4 Aliphatic 3 3 AALLVPAAVLAP 12 57.3 187.5 2.1 Aliphatic 4 4ALALLPVAALAP 12 57.3 195.8 2.1 Aliphatic 5 5 AAALLPVALVAP 12 57.3 187.52.1 Aliphatic 6 11 VVALAPALAALP 12 57.3 187.5 2.1 Aliphatic 7 12LLAAVPAVLLAP 12 57.3 211.7 2.3 Aliphatic 8 13 AAALVPVVALLP 12 57.3 203.32.3 Aliphatic 9 21 AVALLPALLAVP 12 57.3 211.7 2.3 Aliphatic 10 22AVVLVPVLAAAP 12 57.3 195.0 2.4 Aliphatic 11 23 VVLVLPAAAAVP 12 57.3195.0 2.4 Aliphatic 12 24 IALAAPALIVAP 12 50.2 195.8 2.2 Aliphatic 13 25IVAVAPALVALP 12 50.2 203.3 2.4 Aliphatic 14 42 VAALPVVAVVAP 12 57.3186.7 2.4 Aliphatic 15 43 LLAAPLVVAAVP 12 41.3 187.5 2.1 Aliphatic 16 44ALAVPVALLVAP 12 57.3 203.3 2.3 Aliphatic 17 61 VAALPVLLAALP 12 57.3211.7 2.3 Aliphatic 18 62 VALLAPVALAVP 12 57.3 203.3 2.3 Aliphatic 19 63AALLVPALVAVP 12 57.3 203.3 2.3 Aliphatic

TABLE 11 Rigidity/ Sturctural Sequence Flexibility Feature HydropathyResidue ID Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 2064 AIVALPVAVLAP 12 50.2 203.3 2.4 Aliphatic 21 65 IAIVAPVVALAP 12 50.2203.3 2.4 Aliphatic 22 81 AALLPALAALLP 12 57.3 204.2 2.1 Aliphatic 23 82AVVLAPVAAVLP 12 57.3 195.0 2.4 Aliphatic 24 83 LAVAAPLALALP 12 41.3195.8 2.1 Aliphatic 25 84 AAVAAPLLLALP 12 41.3 195.8 2.1 Aliphatic 26 85LLVLPAAALAAP 12 57.3 195.8 2.1 Aliphatic 27 101 LVALAPVAAVLP 12 57.3203.3 2.3 Aliphatic 28 102 LALAPAALALLP 12 57.3 204.2 2.1 Aliphatic 29103 ALIAAPILALAP 12 57.3 204.2 2.2 Aliphatic 30 104 AVVAAPLVLALP 12 41.3203.3 2.3 Aliphatic 31 105 LLALAPAALLAP 12 57.3 204.1 2.1 Aliphatic 32121 AIVALPALALAP 12 50.2 195.8 2.2 Aliphatic 33 123 AAIIVPAALLAP 12 50.2195.8 2.2 Aliphatic 34 124 IAVALPALIAAP 12 50.3 195.8 2.2 Aliphatic 35141 AVIVLPALAVAP 12 50.2 203.3 2.4 Aliphatic 36 143 AVLAVPAVLVAP 12 57.3195.0 2.4 Aliphatic 37 144 VLAIVPAVALAP 12 50.2 203.3 2.4 Aliphatic 38145 LLAVVPAVALAP 12 57.3 203.3 2.3 Aliphatic 39 161 AVIALPALIAAP 12 57.3195.8 2.2 Aliphatic 40 162 AVVALPAALIVP 12 50.2 203.3 2.4 Aliphatic 41163 LALVLPAALAAP 12 57.3 195.8 2.1 Aliphatic 42 164 LAAVLPALLAAP 12 57.3195.8 2.1 Aliphatic 43 165 ALAVPVALAIVP 12 50.2 203.3 2.4 Aliphatic 44182 ALIAPVVALVAP 12 57.3 203.3 2.4 Aliphatic 45 183 LLAAPVVIALAP 12 57.3211.6 2.4 Aliphatic 46 184 LAAIVPAIIAVP 12 50.2 211.6 2.4 Aliphatic 47185 AALVLPLIIAAP 12 41.3 220.0 2.4 Aliphatic 48 201 LALAVPALAALP 12 57.3195.8 2.1 Aliphatic 49 204 LIAALPAVAALP 12 57.3 195.8 2.2 Aliphatic 50205 ALALVPAIAALP 12 57.3 195.8 2.2 Aliphatic 51 221 AAILAPIVALAP 12 50.2195.8 2.2 Aliphatic 52 222 ALLIAPAAVIAP 12 57.3 195.8 2.2 Aliphatic 53223 AILAVPIAVVAP 12 57.3 203.3 2.4 Aliphatic 54 224 ILAAVPIALAAP 12 57.3195.8 2.2 Aliphatic 55 225 VAALLPAAAVLP 12 57.3 187.5 2.1 Aliphatic 56241 AAAVVPVLLVAP 12 57.3 195.0 2.4 Aliphatic 57 242 AALLVPALVAAP 12 57.3187.5 2.1 Aliphatic 58 243 AAVLLPVALAAP 12 57.3 187.5 2.1 Aliphatic 59245 AAALAPVLALVP 12 57.3 187.5 2.1 Aliphatic 60 261 LVLVPLLAAAAP 12 41.3211.6 2.3 Aliphatic 61 262 ALIAVPAIIVAP 12 50.2 211.6 2.4 Aliphatic 62263 ALAVIPAAAILP 12 54.9 195.8 2.2 Aliphatic 63 264 LAAAPVVIVIAP 12 50.2203.3 2.4 Aliphatic 64 265 VLAIAPLLAAVP 12 41.3 211.6 2.3 Aliphatic 65281 ALIVLPAAVAVP 12 50.2 203.3 2.4 Aliphatic 66 282 VLAVAPALIVAP 12 50.2203.3 2.4 Aliphatic 67 283 AALLAPALIVAP 12 50.2 195.8 2.2 Aliphatic 68284 ALIAPAVALIVP 12 50.2 211.7 2.4 Aliphatic 69 285 AIVLLPAAVVAP 12 50.2203.3 2.4 Aliphatic

TABLE 12 Rigidity/ Sturctural Sequence Flexibility Feature HydropathyResidue ID Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 70301 VIAAPVLAVLAP 12 57.3 203.3 2.4 Aliphatic 71 302 LALAPALALLAP 12 57.3204.2 2.1 Aliphatic 72 304 AIILAPIAAIAP 12 57.3 204.2 2.3 Aliphatic 73305 IALAAPILLAAP 12 57.3 204.2 2.2 Aliphatic 74 321 IVAVALPALAVP 12 50.2203.3 2.3 Aliphatic 75 322 VVAIVLPALAAP 12 50.2 203.3 2.3 Aliphatic 76323 IVAVALPVALAP 12 50.2 203.3 2.3 Aliphatic 77 324 IVAVALPAALVP 12 50.2203.3 2.3 Aliphatic 78 325 IVAVALPAVALP 12 50.2 203.3 2.3 Aliphatic 79341 IVAVALPAVLAP 12 50.2 203.3 2.3 Aliphatic 80 342 VIVALAPAVLAP 12 50.2203.3 2.3 Aliphatic 81 343 IVAVALPALVAP 12 50.2 203.3 2.3 Aliphatic 82345 ALLIVAPVAVAP 12 50.2 203.3 2.3 Aliphatic 83 361 AVVIVAPAVIAP 12 50.2195.0 2.4 Aliphatic 84 363 AVLAVAPALIVP 12 50.2 203.3 2.3 Aliphatic 85364 LVAAVAPALIVP 12 50.2 203.3 2.3 Aliphatic 86 365 AVIVVAPALLAP 12 50.2203.3 2.3 Aliphatic 87 381 VVAIVLPAVAAP 12 50.2 195.0 2.4 Aliphatic 88382 AAALVIPAILAP 12 54.9 195.8 2.2 Aliphatic 89 383 VIVALAPALLAP 12 50.2211.6 2.3 Aliphatic 90 384 VIVAIAPALLAP 12 50.2 211.6 2.4 Aliphatic 91385 IVAIAVPALVAP 12 50.2 203.3 2.4 Aliphatic 92 401 AALAVIPAAILP 12 54.9195.8 2.2 Aliphatic 93 402 ALAAVIPAAILP 12 54.9 195.8 2.2 Aliphatic 94403 AAALVIPAAILP 12 54.9 195.8 2.2 Aliphatic 95 404 LAAAVIPAAILP 12 54.9195.8 2.2 Aliphatic 96 405 LAAAVIPVAILP 12 54.9 211.7 2.4 Aliphatic 97421 AAILAAPLIAVP 12 57.3 195.8 2.2 Aliphatic 98 422 VVAILAPLLAAP 12 57.3211.7 2.4 Aliphatic 99 424 AVVVAAPVLALP 12 57.3 195.0 2.4 Aliphatic 100425 AVVAIAPVLALP 12 57.3 203.3 2.4 Aliphatic 101 442 ALAALVPAVLVP 1257.3 203.3 2.3 Aliphatic 102 443 ALAALVPVALVP 12 57.3 203.3 2.3Aliphatic 103 444 LAAALVPVALVP 12 57.3 203.3 2.3 Aliphatic 104 445ALAALVPALVVP 12 57.3 203.3 2.3 Aliphatic 105 461 IAAVIVPAVALP 12 50.2203.3 2.4 Aliphatic 106 462 IAAVLVPAVALP 12 57.3 203.3 2.4 Aliphatic 107463 AVAILVPLLAAP 12 57.3 211.7 2.4 Aliphatic 108 464 AVVILVPLAAAP 1257.3 203.3 2.4 Aliphatic 109 465 IAAVIVPVAALP 12 50.2 203.3 2.4Aliphatic 110 481 AIAIAIVPVALP 12 50.2 211.6 2.4 Aliphatic 111 482ILAVAAIPVAVP 12 54.9 203.3 2.4 Aliphatic 112 483 ILAAAIIPAALP 12 54.9204.1 2.2 Aliphatic 113 484 LAVVLAAPAIVP 12 50.2 203.3 2.4 Aliphatic 114485 AILAAIVPLAVP 12 50.2 211.6 2.4 Aliphatic 115 501 VIVALAVPALAP 1250.2 203.3 2.4 Aliphatic 116 502 AIVALAVPVLAP 12 50.2 203.3 2.4Aliphatic 117 503 AAIIIVLPAALP 12 50.2 220.0 2.4 Aliphatic 118 504LIVALAVPALAP 12 50.2 211.7 2.4 Aliphatic 119 505 AIIIVIAPAAAP 12 50.2195.8 2.3 Aliphatic

TABLE 13 Rigidity/ Sturctural Sequence Flexibility Feature HydropathyResidue ID Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 120521 LAALIVVPAVAP 12 50.2 203.3 2.4 Aliphatic 121 522 ALLVIAVPAVAP 1257.3 203.3 2.4 Aliphatic 122 524 AVALIVVPALAP 12 50.2 203.3 2.4Aliphatic 123 525 ALAIVVAPVAVP 12 50.2 195.0 2.4 Aliphatic 124 541LLALIIAPAAAP 12 57.3 204.1 2.1 Aliphatic 125 542 ALALIIVPAVAP 12 50.2211.6 2.4 Aliphatic 126 543 LLAALIA^(P)AAL^(P) 12 57.3 204.1 2.1Aliphatic 127 544 IVALIVAPAAVP 12 43.1 203.3 2.4 Aliphatic 128 545VVLVLAAPAAVP 12 57.3 195.0 2.3 Aliphatic 129 561 AAVAIVLPAVVP 12 50.2195.0 2.4 Aliphatic 130 562 ALIAAIVPALVP 12 50.2 211.7 2.4 Aliphatic 131563 ALAVIVVPALAP 12 50.2 203.3 2.4 Aliphatic 132 564 VAIALIVPALAP 1250.2 211.7 2.4 Aliphatic 133 565 VAIVLVAPAVAP 12 50.2 195.0 2.4Aliphatic 134 582 VAVALIVPALAP 12 50.2 203.3 2.4 Aliphatic 135 583AVILALAPIVAP 12 50.2 211.6 2.4 Aliphatic 136 585 ALIVAIAPALVP 12 50.2211.6 2.4 Aliphatic 137 601 AAILIAVPIAAP 12 57.3 195.8 2.3 Aliphatic 138602 VIVALAAPVLAP 12 50.2 203.3 2.4 Aliphatic 139 603 VLVALAAPVIAP 1257.3 203.3 2.4 Aliphatic 140 604 VALIAVAPAVVP 12 57.3 195.0 2.4Aliphatic 141 605 VIAAVLAPVAVP 12 57.3 195.0 2.4 Aliphatic 142 622ALIVLAAPVAVP 12 50.2 203.3 2.4 Aliphatic 143 623 VAAAIALPAIVP 12 50.2187.5 2.3 Aliphatic 144 625 ILAAAAAPLIVP 12 50.2 195.8 2.2 Aliphatic 145643 LALVLAAPAIVP 12 50.2 211.6 2.4 Aliphatic 146 645 ALAVVALPAIVP 1250.2 203.3 2.4 Aliphatic 147 661 AAILAPIVAALP 12 50.2 195.8 2.2Aliphatic 148 664 ILIAIAIPAAAP 12 54.9 204.1 2.3 Aliphatic 149 665LAIVLAAPVAVP 12 50.2 203.3 2.3 Aliphatic 150 666 AAIAIIAPAIVP 12 50.2195.8 2.3 Aliphatic 151 667 LAVAIVAPALVP 12 50.2 203.3 2.3 Aliphatic 152683 LAIVLAAPAVLP 12 50.2 211.7 2.4 Aliphatic 153 684 AAIVLALPAVLP 1250.2 211.7 2.4 Aliphatic 154 685 ALLVAVLPAALP 12 57.3 211.7 2.3Aliphatic 155 686 AALVAVLPVALP 12 57.3 203.3 2.3 Aliphatic 156 687AILAVALPLLAP 12 57.3 220.0 2.3 Aliphatic 157 703 IVAVALVPALAP 12 50.2203.3 2.4 Aliphatic 158 705 IVAVALLPALAP 12 50.2 211.7 2.4 Aliphatic 159706 IVAVALLPAVAP 12 50.2 203.3 2.4 Aliphatic 160 707 IVALAVLPAVAP 1250.2 203.3 2.4 Aliphatic 161 724 VAVLAVLPALAP 12 57.3 203.3 2.3Aliphatic 162 725 IAVLAVAPAVLP 12 57.3 203.3 2.3 Aliphatic 163 726LAVAIIAPAVAP 12 57.3 187.5 2.2 Aliphatic 164 727 VALAIALPAVLP 12 57.3211.6 2.3 Aliphatic 165 743 AIAIALVPVALP 12 57.3 211.6 2.4 Aliphatic 166744 AAVVIVAPVALP 12 50.2 195.0 2.4 Aliphatic 167 746 VAIIVVAPALAP 1250.2 203.3 2.4 Aliphatic 168 747 VALLAIAPALAP 12 57.3 195.8 2.2Aliphatic 169 763 VAVLIAVPALAP 12 57.3 203.3 2.3 Aliphatic

TABLE 14 Rigidity/ Sturctural Sequence Flexibility Feature HydropathyResidue ID Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 170764 AVALAVLPAVVP 12 57.3 195.0 2.3 Aliphatic 171 765 AVALAVVPAVLP 1257.3 195.0 2.3 Aliphatic 172 766 IVVIAVAPAVAP 12 50.2 195.0 2.4Aliphatic 173 767 IVVAAVVPALAP 12 50.2 195.0 2.4 Aliphatic 174 783IVALVPAVAIAP 12 50.2 203.3 2.5 Aliphatic 175 784 VAALPAVALVVP 12 57.3195.0 2.4 Aliphatic 176 786 LVAIAPLAVLAP 12 41.3 211.7 2.4 Aliphatic 177787 AVALVPVIVAAP 12 50.2 195.0 2.4 Aliphatic 178 788 AIAVAIAPVALP 1257.3 187.5 2.3 Aliphatic 179 803 AIALAVPVLALP 12 57.3 211.7 2.4Aliphatic 180 805 LVLIAAAPIALP 12 41.3 220.0 2.4 Aliphatic 181 806LVALAVPAAVLP 12 57.3 203.3 2.3 Aliphatic 182 807 AVALAVPALVLP 12 57.3203.3 2.3 Aliphatic 183 808 LVVLAAAPLAVP 12 41.3 203.3 2.3 Aliphatic 184809 LIVLAAPALAAP 12 50.2 195.8 2.2 Aliphatic 185 810 VIVLAAPALAAP 1250.2 187.5 2.2 Aliphatic 186 811 AVVLAVPALAVP 12 57.3 195.0 2.3Aliphatic 187 824 LIIVAAAPAVAP 12 50.2 187.5 2.3 Aliphatic 188 825IVAVIVAPAVAP 12 43.2 195.0 2.5 Aliphatic 189 826 LVALAAPIIAVP 12 41.3211.7 2.4 Aliphatic 190 827 IAAVLAAPALVP 12 57.3 187.5 2.2 Aliphatic 191828 IALLAAPIIAVP 12 41.3 220.0 2.4 Aliphatic 192 829 AALALVAPVIVP 1250.2 203.3 2.4 Aliphatic 193 830 IALVAAPVALVP 12 57.3 203.3 2.4Aliphatic 194 831 IIVAVAPAAIVP 12 43.2 203.3 2.5 Aliphatic 195 832AVAAIVPVIVAP 12 43.2 195.0 2.5 Aliphatic 196 843 AVLVLVAPAAAP 12 41.3219.2 2.5 Aliphatic 197 844 VVALLAPLIAAP 12 41.3 211.8 2.4 Aliphatic 198845 AAVVIAPLLAVP 12 41.3 203.3 2.4 Aliphatic 199 846 IAVAVAAPLLVP 1241.3 203.3 2.4 Aliphatic 200 847 LVAIVVLPAVAP 12 50.2 219.2 2.6Aliphatic 201 848 AVAIVVLPAVAP 12 50.2 195.0 2.4 Aliphatic 202 849AVILLAPLIAAP 12 57.3 220.0 2.4 Aliphatic 203 850 LVIALAAPVALP 12 57.3211.7 2.4 Aliphatic 204 851 VLAVVLPAVALP 12 57.3 219.2 2.5 Aliphatic 205852 VLAVAAPAVLLP 12 57.3 203.3 2.3 Aliphatic 206 863 AAVVLLPIIAAP 1241.3 211.7 2.4 Aliphatic 207 864 ALLVIAPAIAVP 12 57.3 211.7 2.4Aliphatic 208 865 AVLVIAVPAIAP 12 57.3 203.3 2.5 Aliphatic 209 867ALLVVIAPLAAP 12 41.3 211.7 2.4 Aliphatic 210 868 VLVAAILPAAIP 12 54.9211.7 2.4 Aliphatic 211 870 VLVAAVLPIAAP 12 41.3 203.3 2.4 Aliphatic 212872 VLAAAVLPLVVP 12 41.3 219.2 2.5 Aliphatic 213 875 AIAIVVPAVAVP 1250.2 195.0 2.4 Aliphatic 214 877 VAIIAVPAVVAP 12 57.3 195.0 2.4Aliphatic 215 878 IVALVAPAAVVP 12 50.2 195.0 2.4 Aliphatic 216 879AAIVLLPAVVVP 12 50.2 219.1 2.5 Aliphatic 217 881 AALIVVPAVAVP 12 50.2195.0 2.4 Aliphatic 218 882 AIALVVPAVAVP 12 57.3 195.0 2.4 Aliphatic 219883 LAIVPAAIAALP 12 50.2 195.8 2.2 Aliphatic

TABLE 15 Rigidity/ Sturctural Sequence Flexibility Feature HydropathyResidue ID Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 220885 LVAIAPAVAVLP 12 57.3 203.3 2.4 Aliphatic 221 887 VLAVAPAVAVLP 1257.3 195.0 2.4 Aliphatic 222 888 ILAVVAIPAAAP 12 54.9 187.5 2.3Aliphatic 223 889 ILVAAAPIAALP 12 57.3 195.8 2.2 Aliphatic 224 891ILAVAAIPAALP 12 54.9 195.8 2.2 Aliphatic 225 893 VIAIPAILAAAP 12 54.9195.8 2.3 Aliphatic 226 895 AIIIVVPAIAAP 12 50.2 211.7 2.5 Aliphatic 227896 AILIVVAPIAAP 12 50.2 211.7 2.5 Aliphatic 228 897 AVIVPVAIIAAP 1250.2 203.3 2.5 Aliphatic 229 899 AVVIALPAVVAP 12 57.3 195.0 2.4Aliphatic 230 900 ALVAVIAPVVAP 12 57.3 195.0 2.4 Aliphatic 231 901ALVAVLPAVAVP 12 57.3 195.0 2.4 Aliphatic 232 902 ALVAPLLAVAVP 12 41.3203.3 2.3 Aliphatic 233 904 AVLAVVAPVVAP 12 57.3 186.7 2.4 Aliphatic 234905 AVIAVAPLVVAP 12 41.3 195.0 2.4 Aliphatic 235 906 AVIALAPVVVAP 1257.3 195.0 2.4 Aliphatic 236 907 VAIALAPVVVAP 12 57.3 195.0 2.4Aliphatic 237 908 VALALAPVVVAP 12 57.3 195.0 2.3 Aliphatic 238 910VAALLPAVVVAP 12 57.3 195.0 2.3 Aliphatic 239 911 VALALPAVVVAP 12 57.3195.0 2.3 Aliphatic 240 912 VALLAPAVVVAP 12 57.3 195.0 2.3 Aliphatic52.6 ± 5.1 201.7 ± 7.8 2.3 ± 0.1

3-4. Design of the Peptides that Did not Satisfy at Least One CriticalFactor

To demonstrate that this invention of new hydrophobic CPPs—aMTDs, whichsatisfy all critical factors described above, are correct and rationallydesigned, the peptides which do not satisfy at least one critical factorhave also been designed. Total of 31 rPeptides (rPs) are designed,developed and categorized as follows: no bending peptides, either noproline in the middle as well at the end and/or no central proline;rigid peptides (II<40); too much flexible peptides; aromatic peptides(aromatic ring presences); hydrophobic, with non-aromatic peptides buthave amino acids other than A, V, L, I, P or additional prolineresidues; hydrophilic, but non-aliphatic peptides.

3-4-1. Peptides that do not Satisfy the Bending Potential

Table 16 shows the peptides that do not have any proline in the middle(at 5′, 6′, 7′ or 8′) and at the end of the sequences. In addition,Table 16 describes the peptides that do not have proline in the middleof the sequences. All these peptides are supposed to have no-bendingpotential.

TABLE 16 SEQ  Proline Rigidity/ Sturctural ID  rPeptide PositionFlexibility Feature Hydropathy Group NO ID Sequences Length (PP) (II)(AI) (GRAVY) No-Bending 882 931 AVLIAPAILAAA 12 6 57.3 204.2 2.5Peptides 884 936 ALLILAAAVAAP 12 12 41.3 204.2 2.4 (No Proline 885 152LAAAVAAVAALL 12 None 9.2 204.2 2.7 at 5,  886 27 LAIVAAAAALVA 12 None2.1 204.2 2.8 6, 7 or 8 887 935 ALLILPAAAVAA 12 6 57.3 204.2 2.4and/or 12) 888 670 ALLILAAAVAAL 12 None 25.2 236.6 2.8 888 934LILAPAAVVAAA 12 5 57.3 195.8 2.5 889 37 TTCSQQQYCTNG 12 None 53.1 0.0−1.1 890 16 NNSCTTYTNGSQ 12 None 47.4 0.0 −1.4 891 113 PVAVALLIAVPP 121,11,12 57.3 195 2.1

3-4-2. Peptides that do not Satisfy the Rigidity/Flexibility

To prove that rigidity/flexibility of the sequence is a crucial criticalfactor, rigid (Avg. II: 21.8±6.6) and too high flexible sequences (Avg.II: 82.3±21.0) were also designed. Rigid peptides that instability indexis much lower than that of new aMTDs (II: 41.3-57.3, Avg. II: 53.3±5.7)are shown in Table 17. Bending, but too high flexible peptides that IIis much higher than that of new aMTDs are also provided in Table 18.

TABLE 17 SEQ Proline Rigidity/ Sturctural ID rPeptide PositionFlexibility Feature Hydropathy Group NO ID Sequences Length (PP) (II)(AI) (GRAVY) Rigid 892 226 ALVAAIPALAIP 12 6 20.4 195.8 2.2 Peptides 8936 VIAMIPAAFWVA 12 6 15.7 146.7 2.2 (II < 50) 894 750 LAIAAIAPLAIP 128,12 22.8 204.2 2.2 895 26 AAIALAAPLAIV 12 8 18.1 204.2 2.5 896 527LVLAAVAPIAIP 12 8,12 22.8 211.7 2.4 897 466 IIAAAAPLAIIP 12 7,12 22.8204.2 2.3 898 167 VAIAIPAALAIP 12 6,12 20.4 195.8 2.3 899 246VVAVPLLVAFAA 12 5 25.2 195 2.7 900 426 AAALAIPLAIIP 12 7,12 4.37 204.22.2 901 606 AAAIAAIPIIIP 12 8,12 4.4 204.2 2.4 902 66 AGVLGGPIMGVP 127,12 35.5 121.7 1.3 903 248 VAAIVPIAALVP 12 6,12 34.2 203.3 2.5 904 227LAAIVPIAAAVP 12 6,12 34.2 187.5 2.2 905 17 GGCSAPQTTCSN 12 6 51.6 8.3−0.5 906 67 LDAEVPLADDVP 12 6,12 34.2 130 0.3

TABLE 18 Proline Rigidity/ Sturctural  SEQ rPeptide Position FlexibilityFeature Hydropathy Group ID NO ID Sequences Length (PP) (II) (AI)(GRAVY) Bending 907 692 PAPLPPVVILAV 12 1,3,5,6 105.5 186.7 1.8Peptides, 908 69 PVAVLPPAALVP 12 1,6,7,12 89.4 162.5 1.6 but Too 909 390VPLLVPVVPVVP 12 2,6,9,12 105.4 210 2.2 High 910 350 VPILVPVVPVVP 122,6,9,12 121.5 210 2.2 Flexibility 911 331 VPVLVPLVPVVP 12 2,6,9,12105.4 210 2.2 912 9 VALVPAALILPP 12 5,11,12 89.4 203.3 2.1 913 68VAPVLPAAPLVP 12 3,6,9,12 105.5 162.5 1.6 914 349 VPVLVPVVPVVP 122,6,9,12 121.5 201.6 2.2 915 937 VPVLVPLPVPVV 12 2,6,8,10 121.5 210 2.2916 938 VPVLLPVVVPVP 12 2,6,10,12 121.5 210 2.2 917 329 LPVLVPVVPVVP 122,6,9,12 121.5 210 2.2 918 49 VVPAAPAVPVVP 12 3,6,9,12 121.5 145.8 1.7919 772 LPVAPVIPIIVP 12 2,5,8,12 79.9 210.8 2.1 920 210 ALIALPALPALP 126,9,12 89.4 195.8 1.8 921 28 AVPLLPLVPAVP 12 3,6,9,12 89.4 186.8 1.8 922693 AAPVLPVAVPIV 12 3,6,10 82.3 186.7 2.1 923 169 VALVAPALILAP 12 6,1273.4 211.7 2.4 924 29 VLPPLPVLPVLP 12 3,4,6,9,12 121.5 202.5 1.7 925 190AAILAPAVIAPP 12 6,11,12 89.4 163.3 1.8

3-4-3. Peptides that do not Satisfy the Structural Features

New hydrophobic CPPs—aMTDs are consisted with only hydrophobic andaliphatic amino acids (A, V, L, I and P) with average ranges of theindexes—AI: 180-220 and GRAVY: 2.1-2.6 (Table 9). Based on thestructural indexes, the peptides which contain an aromatic residue (W, For Y) are shown in Table 19 and the peptides which are hydrophobic withnon-aromatic sequences but have amino acids residue other than A, V, L,I, P or additional proline residues are designed (Table 20). Finally,hydrophilic and/or bending peptides which are consisted withnon-aliphatic amino acids are shown in Table 21.

TABLE 19 SEQ Proline Rigidity/ Sturctural ID rPeptide PositionFlexibility Feature Hydropathy Group NO ID Sequences Length (PP) (II)(AI) (GRAVY) Aromatic 926 30 WFFAGPIMLIWP 12 6, 12 9.2 105.8 1.4Peptides 927 33 AAAILAPAFLAV 12 7 57.3 171.7 2.4 (Aromatic 928 131WIIAPVWLAWIA 12 5 51.6 179.2 1.9 Ring 929 922 WYVIFVLPLVVP 12 8,12 41.3194.2 2.2 Presences) 930 71 FMWMWFPFMWYP 12 7,12 71.3 0.0 0.6 931 921IWWFVVLPLVVP 12 8,12 41.3 194.2 2.2

TABLE 20 SEQ Proline Rigidity/ Sturctural ID rPeptide PositionFlexibility Feature Hydropathy Group NO ID Sequences Length (PP) (II)(AI) (GRAVY) Hydrophobic 932 436 VVMLVVPAVMLP 12 7,12 57.3 194.2 2.6but Non 933 138 PPAALLAILAVA 12 1,2 57.3 195.8 2.2 Aromatic 934 77PVALVLVALVAP 12 1,12 41.3 219.2 2.5 Peptides 935 577 MLMIALVPMIAV 12 818.9 195.0 2.7 936 97 ALLAAPPALLAL 12 6,7 57.3 204.2 2.1 937 214ALIVAPALMALP 12 6,12 60.5 187.5 2.2 938 59 AVLAAPVVAALA 12 6 41.3 187.52.5 939 54 LAVAAPPVVALL 12 6,7 57.3 203.3 2.3

TABLE 21 SEQ Proline Rigidity/ Sturctural ID rPeptide PositionFlexibility Feature Hydropathy Group NO ID Sequences Length (PP) (II)(AI) (GRAVY) Hydrophilic 940 949 SGNSCQQCGNSS 12 None 41.7 0.0 -1.1Peptides 941 39 CYNTSPCTGCCY 12 6 52.5 0.0 0.0 but Non 942 19YVSCCTYTNGSQ 12 None 47.7 0.0 -1.0 Aliphatic 943 947 CYYNQQSNNNNQ 12None 59.6 0.0 -2.4 944 139 TGSTNSPTCTST 12 7 53.4 0.0 -0.7 945 18NYCCTPTTNGQS 12 6 47.9 0.0 -0.9 946 20 NYCNTCPTYGQS 12 7 47.4 0.0 -0.9947 635 GSTGGSQQNNQY 12 None 31.9 0.0 -1.9 948 40 TYNTSCTPGTCY 12 8 49.40,0 -0.6 949 57 QNNCNTSSQGGG 12 None 52.4 0.0 -1.6 950 159 CYSGSTSQNQPP12 11,12 51.0 0.0 -1.3 951 700 GTSNTCQSNQNS 12 None 19.1 0.0 -1.6 952 38YYNQSTCGGQCY 12 None 53.8 0.0 -1.0

3-5. Summary of Newly Designed Peptides

Total of 457 sequences have been designed based on the critical factors.Designed potentially best aMTDs (hydrophobic, flexible, bending,aliphatic and 12-A/a length peptides) that do satisfy all range/featureof critical factors are 316. Designed rPeptides that do not satisfy atleast one of the critical factors are 141 that no bending peptidesequences are 26; rigid peptide (II<40) sequences are 23; too muchflexible peptides are 24; aromatic peptides (aromatic ring presences)are 27; hydrophobic, but non-aromatic peptides are 23; and hydrophilic,but non-aliphatic peptides are 18.

4. Preparation of Recombinant Report Proteins Fused to aMTDs andrPeptides

Recombinant proteins fused to aMTDs and others [rPeptides, referencehydrophobic CPP sequences (MTM and MTD)] were expressed in a bacterialsystem, purified with single-step affinity chromatography and preparedas soluble proteins in physiological condition. These recombinantproteins have been tested for the ability of their cell-permeability byutilizing flow cytometry and laser scanning confocal microscopy.

4-1. Selection of Cargo Protein for Recombinant Proteins Fused toPeptide Sequences

For clinical/non-clinical application, aMTD-fused cargo materials wouldbe biologically active molecules that could be one of the following:enzymes, transcription factors, toxic, antigenic peptides, antibodiesand antibody fragments. Furthermore, biologically active molecules couldbe one of these following macromolecules: enzymes, hormones, carriers,immunoglobulin, membrane-bound proteins, transmembrane proteins,internal proteins, external proteins, secreted proteins, virus proteins,native proteins, glycoproteins, fragmented proteins, disulfide bondedproteins, recombinant proteins, chemically modified proteins and prions.In addition, these biologically active molecules could be one of thefollowing: nucleic acid, coding nucleic acid sequence, mRNAs, antisenseRNA molecule, carbohydrate, lipid and glycolipid.

According to these pre-required conditions, a non-functional cargo toevaluate aMTD-mediated protein uptake has been selected and called asCargo A (CRA) that should be soluble and non-functional. The domain (A/a289-840; 184 A/a length) is derived from protein S (Genbank ID:CP000113.1).

4-2. Construction of Expression Vector and Preparation of RecombinantProteins

Coding sequences for recombinant proteins fused to each aMTD are clonedNde1 (5′) and Sail (3′) in pET-28a(+) (Novagen, Darmstadt, Germany) fromPCR-amplified DNA segments. PCR primers for the recombinant proteinsfused to aMTD and rPeptides are SEQ ID NOs: 481-797. Structure of therecombinant proteins is displayed in FIG. 1.

The recombinant proteins were forcedly expressed in E. coli BL21 (DE3)cells grown to an OD₆₀₀ of 0.6 and induced for 2 hours with 0.7 mMisopropyl-β-D-thiogalactopyranoside (IPTG). The proteins were purifiedby Ni²⁺ affinity chromatography as directed by the supplier (Qiagen,Hilden, Germany) in natural condition. After the purification, purifiedproteins were dissolved in a physiological buffer such as DMEM medium.

TABLE 22 Potentially Best aMTDs (Hydrophobic, Flexible, 240 Bending,Aliphatic & Helical) Random Peptides Total 31 No Bending Peptides (NoProline at 5 or 6 and/or 12) 02 No Bending Peptides (No Central Proline)01 Rigid Peptides (II < 50) 09 Too Much Flexible Peptides 09 AromaticPeptides (Aromatic Ring Presences) 01 Hydrophobic, But Non-AromaticPeptides 02 Hydrophilic, But Non-Aliphatic Peptides 07

4-3. Expression of aMTD- or Random Peptide (rP)-Fused RecombinantProteins

Using the standardized six critical factors, 316 aMTD sequences havebeen designed. In addition, 141 rPeptides are also developed that lackone of these critical factors: no bending peptides: i) absence ofproline both in the middle and at the end of sequence or ii) absence ofproline either in the middle or at the end of sequence, rigid peptides,too much flexible peptides, aromatic peptides (aromatic ring presence),hydrophobic but non-aromatic peptides, and hydrophilic but non-aliphaticpeptides (Table 22).

These rPeptides are devised to be compared and contrasted with aMTDs inorder to analyze structure/sequence activity relationship (SAR) of eachcritical factor with regard to the peptides' intracellular deliverypotential. All peptide (aMTD or rPeptide)-containing recombinantproteins have been fused to the CRA to enhance the solubility of therecombinant proteins to be expressed, purified, prepared and analyzed.

These designed 316 aMTDs and 141 rPeptides fused to CRA were all cloned(FIG. 2) and tested for inducible expression in E. coli (FIG. 3). Out ofthese peptides, 240 aMTDs were inducibly expressed, purified andprepared in soluble form (FIG. 4). In addition, 31 rPeptides were alsoprepared as soluble form (FIG. 4).

To prepare the proteins fused to rPeptides, 60 proteins were expressedthat were 10 out of 26 rPeptides in the category of no bending peptides(Table 16); 15 out of 23 in the category of rigid peptides [instabilityindex (II)<40] (Table 17); 19 out of 24 in the category of too muchflexible peptides (Table 18); 6 out of 27 in the category of aromaticpeptides (Table 19); 8 out of 23 in the category of hydrophobic butnon-aromatic peptides (Table 20); and 12 out of 18 in the category ofhydrophilic but non-aliphatic peptides (Table 21).

4-4. Quantitative Cell-Permeability of aMTD-Fused Recombinant Proteins

The aMTDs and rPeptides were fluorescently labeled and compared based onthe critical factors for cell-permeability by using flow cytometry andconfocal laser scanning microscopy (FIGS. 5 to 8). The cellular uptakeof the peptide-fused non-functional cargo recombinant proteins couldquantitatively be evaluated in flow cytometry, while confocal laserscanning microscopy allows intracellular uptake to be assessed visually.The analysis included recombinant proteins fused to a negative control[rP38] that has opposite characteristics (hydrophilic and aromaticsequence: YYNQSTCGGQCY (SEQ ID NO: 952)) to the aMTDs (hydrophobic andaliphatic sequences). Relative cell-permeability (relative fold) ofaMTDs to the negative control was also analyzed (Table 23 and FIG. 9).

Table 23 shows Comparison Analysis of Cell-Permeability of aMTDs with aNegative Control (A: rP38).

TABLE 23 Negative Control rP38 aMTD 19.6 ± 1.6* The Average of 240 aMTDs(Best: 164.2) *Relative Fold (aMTD in Geo Mean in its comparison torP38)

Relative cell-permeability (relative fold) of aMTDs to the referenceCPPs [B: MTM12 (AAVLLPVLLAAP)(SEQ ID NO: 953), C: MTD85 (AVALLILAV) (SEQID NO: 954)] was also analyzed (Tables 40 and 41)

Table 24 shows Comparison Analysis of Cell-Permeability of aMTDs with aReference CPP (B: MTM12).

TABLE 24 MTM12 aMTD 13.1 ± 1.1* The Average of 240 aMTDs (Best: 109.9)*Relative Fold (aMTD in Geo Mean in its comparison to MTM12)

Table 25 shows Comparison Analysis of Cell-Permeability of aMTDs with aReference CPP (C: MTD85).

TABLE 25 MTD85 aMTD 6.6 ± 0.5* The Average of 240 aMTDs (Best: 55.5)*Relative Fold (aMTD in Geo Mean in its comparison to MTD85)

Geometric means of negative control (histidine-tagged rP38-fused CRArecombinant protein) subtracted by that of naked protein(histidine-tagged CRA protein) lacking any peptide (rP38 or aMTD) wasstandardized as relative fold of 1. Relative cell-permeability of 240aMTDs to the negative control (A type) was significantly increased by upto 164 fold, with average increase of 19.6±1.6 (Table 26-31).

TABLE 26 SEQ Proline Rigidity/ Sturctural Relative Ratio ID PositionFlexibility Feature Hydropathy (Fold) NO aMTD Sequences Length (PP) (II)(AI) (GRAVY) A B C 229 899 AVVIALPAVVAP 12 7 57.3 195.0 2.4 164.2 109.955.5 237 908 VALALAPVVVAP 12 7 57.3 195.0 2.3 150.6 100.8 50.9 238 910VAALLPAVVVAP 12 6 57.3 195.0 2.3 148.5 99.4 50.2 185 810 VIVLAAPALAAP 127 50.2 187.5 2.2 120.0 80.3 40.6 233 904 AVLAVVAPVVAP 12 8 57.3 186.72.4 105.7 70.8 35.8 74 321 IVAVALPALAVP 12 7 50.2 203.3 2.3 97.8 65.232.9 204 851 VLAVVLPAVALP 12 7 57.3 219.2 2.5 96.6 64.7 32.7 239 911VALALPAVVVAP 12 6 57.3 195.0 2.3 84.8 56.8 28.7 205 852 VLAVAAPAVLLP 127 57.3 203.3 2.3 84.6 56.6 28.6 179 803 AIALAVPVLALP 12 7 57.3 211.7 2.474.7 50.0 25.3 222 888 ILAVVAIPAAAP 12 8 54.9 187.5 2.3 71.0 47.5 24.0188 825 IVAVIVAPAVAP 12 8 43.2 195.0 2.5 69.7 46.6 23.6 226 895AIIIVVPAIAAP 12 7 50.2 211.7 2.5 60.8 40.7 20.6 227 896 AILIVVAPIAAP 128 50.2 211.7 2.5 57.5 38.5 19.4 164 727 VALAIALPAVLP 12 8 57.3 211.6 2.354.7 36.7 18.5 139 603 VLVALAAPVIAP 12 8 57.3 203.3 2.4 54.1 36.1 18.2200 847 LVAIVVLPAVAP 12 8 50.2 219.2 2.6 50.2 33.4 16.9 189 826LVALAAPIIAVP 12 7 41.3 211.7 2.4 49.2 32.9 16.6 161 724 VAVLAVLPALAP 128 57.3 203.3 2.3 47.5 31.8 16.1 131 563 ALAVIVVPALAP 12 8 50.2 203.3 2.447.1 31.4 15.9 186 811 AVVLAVPALAVP 12 7 57.3 195.0 2.3 46.5 31.1 15.7194 831 IIVAVAPAAIVP 12 7 43.2 203.3 2.5 46.3 31.0 15.7 192 829AALALVAPVIVP 12 8 50.2 203.3 2.4 44.8 30.0 15.2 224 891 ILAVAAIPAALP 128 54.9 195.8 2.2 44.7 29.9 15.1 234 905 AVIAVAPLVVAP 12 7 41.3 195.0 2.444.0 29.5 14.9 132 564 VAIALIVPALAP 12 8 50.2 211.7 2.4 43.6 29.1 14.734 124 IAVALPALIAAP 12 6 50.3 195.8 2.2 43.6 29.0 14.7 190 827IAAVLAAPALVP 12 8 57.3 187.5 2.2 43.0 28.8 14.6 2 2 AAAVPLLAVVVP 12 541.3 195.0 2.4 40.9 27.2 13.8 91 385 IVAIAVPALVAP 12 7 50.2 203.3 2.438.8 25.9 13.1 191 828 IALLAAPIIAVP 12 7 41.3 220.0 2.4 36.8 24.6 12.4181 806 LVALAVPAAVLP 12 7 57.3 203.3 2.3 36.7 24.6 12.4 198 845AAVVIAPLLAVP 12 7 41.3 203.3 2.4 35.8 24.0 12.1 218 882 AIALVVPAVAVP 127 57.3 195.0 2.4 35.0 23.4 11.8 128 545 VVLVLAAPAAVP 12 8 57.3 195.0 2.334.6 23.1 11.7 39 161 AVIALPALIAAP 12 6 57.3 195.8 2.2 34.5 23.0 11.6110 481 AIAIAIVPVALP 12 8 50.2 211.6 2.4 34.3 23.0 11.6 230 900ALVAVIAPVVAP 12 8 57.3 195.0 2.4 34.3 22.9 11.6 53 223 AILAVPIAVVAP 12 657.3 203.3 2.4 33.0 22.1 11.2 187 824 LIIVAAAPAVAP 12 8 50.2 187.5 2.332.8 21.9 11.1 130 562 ALIAAIVPALVP 12 8 50.2 211.7 2.4 32.7 21.8 11.052 222 ALLIAPAAVIAP 12 6 57.3 195.8 2.2 32.6 21.7 11.0 17 61VAALPVLLAALP 12 5 57.3 211.7 2.3 31.2 20.8 10.5 134 582 VAVALIVPALAP 128 50.2 203.3 2.4 30.6 20.4 10.3 223 889 ILVAAAPIAALP 12 7 57.3 195.8 2.230.3 20.3 10.3 177 787 AVALVPVIVAAP 12 6 50.2 195.0 2.4 29.3 19.6 9.9157 703 IVAVALVPALAP 12 8 50.2 203.3 2.4 29.2 19.5 9.9 158 705IVAVALLPALAP 12 8 50.2 211.7 2.4 28.6 19.1 9.7 220 885 LVAIAPAVAVLP 12 657.3 203.3 2.4 28.3 19.0 9.6 3 3 AALLVPAAVLAP 12 6 57.3 187.5 2.1 27.018.0 9.1 137 601 AAILIAVPIAAP 12 8 57.3 195.8 2.3 26.8 17.9 9.0 196 843AVLVLVAPAAAP 12 8 41.3 219.2 2.5 26.4 17.7 8.9 94 403 AAALVIPAAILP 12 754.9 195.8 2.2 25.2 16.8 8.5 127 544 IVALIVAPAAVP 12 8 43.1 203.3 2.423.4 15.6 7.9 121 522 ALLVIAVPAVAP 12 8 57.3 203.3 2.4 22.7 15.2 7.7

TABLE 27 SEQ Proline Rigidity/ Sturctural Relative Ratio ID PositionFlexibility Feature Hydropathy (Fold) NO aMTD Sequences Length (PP) (II)(AI) (GRAVY) A B C 180 805 LVLIAAAPIALP 12 8 41.3 220.0 2.4 22.3 14.97.6 108 464 AVVILVPLAAAP 12 7 57.3 203.3 2.4 22.3 14.9 7.5 96 405LAAAVIPVAILP 12 7 54.9 211.7 2.4 22.2 14.8 7.5 168 747 VALLAIAPALAP 12 857.3 195.8 2.2 22.0 14.8 7.5 115 501 VIVALAVPALAP 12 8 50.2 203.3 2.421.5 14.4 7.3 147 661 AAILAPIVAALP 12 6 50.2 195.8 2.2 21.4 14.3 7.2 176786 LVAIAPLAVLAP 12 6 41.3 211.7 2.4 21.2 14.2 7.2 144 625 ILAAAAAPLIVP12 8 50.2 195.8 2.2 20.9 13.9 7.0 101 442 ALAALVPAVLVP 12 7 57.3 203.32.3 20.4 13.6 6.9 240 912 VALLAPAVVVAP 12 6 57.3 195.0 2.3 19.9 13.3 6.743 165 ALAVPVALAIVP 12 5 50.2 203.3 2.4 19.8 13.2 6.7 98 422VVAILAPLLAAP 12 7 57.3 211.7 2.4 19.6 13.1 6.6 155 686 AALVAVLPVALP 12 857.3 203.3 2.3 19.5 13.1 6.6 81 343 IVAVALPALVAP 12 7 50.2 203.3 2.319.4 12.9 6.5 76 323 IVAVALPVALAP 12 7 50.2 203.3 2.3 19.1 12.8 6.4 105461 IAAVIVPAVALP 12 7 50.2 203.3 2.4 19.0 12.7 6.4 9 21 AVALLPALLAVP 126 57.3 211.7 2.3 18.9 12.6 6.4 95 404 LAAAVIPAAILP 12 7 54.9 195.8 2.218.9 12.6 6.4 60 261 LVLVPLLAAAAP 12 5 41.3 211.6 2.3 18.5 12.3 6.2 122524 AVALIVVPALAP 12 8 50.2 203.3 2.4 18.3 12.2 6.2 55 225 VAALLPAAAVLP12 6 57.3 187.5 2.1 18.3 12.2 6.2 63 264 LAAAPVVIVIAP 12 5 50.2 203.32.4 18.2 12.1 6.1 1 1 AAALAPVVLALP 12 6 57.3 187.5 2.1 17.7 11.8 6.0 88382 AAALVIPAILAP 12 7 54.9 195.8 2.2 17.7 11.8 6.0 107 463 AVAILVPLLAAP12 7 57.3 211.7 2.4 17.6 11.7 5.9 75 322 VVAIVLPALAAP 12 7 50.2 203.32.3 17.6 11.7 5.9 117 503 AAIIIVLPAALP 12 8 50.2 220.0 2.4 17.6 11.8 5.9211 870 VLVAAVLPIAAP 12 8 41.3 203.3 2.4 16.6 11.1 5.6 56 241AAAVVPVLLVAP 12 6 57.3 195.0 2.4 16.6 11.0 5.6 163 726 LAVAIIAPAVAP 12 857.3 187.5 2.2 16.5 11.0 5.6 79 341 IVAVALPAVLAP 12 7 50.2 203.3 2.316.4 10.9 5.5 125 542 ALALIIVPAVAP 12 8 50.2 211.6 2.4 16.2 10.8 5.5 83361 AVVIVAPAVIAP 12 7 50.2 195.0 2.4 16.0 10.7 5.4 54 224 ILAAVPIALAAP12 6 57.3 195.8 2.2 15.8 10.6 5.3 20 64 AIVALPVAVLAP 12 6 50.2 203.3 2.415.8 10.6 5.3 111 482 ILAVAAIPVAVP 12 8 54.9 203.3 2.4 15.8 10.6 5.3 113484 LAVVLAAPAIVP 12 8 50.2 203.3 2.4 15.6 10.4 5.3 210 868 VLVAAILPAAIP12 8 54.9 211.7 2.4 14.9 10.0 5.0 124 541 LLALIIAPAAAP 12 8 57.3 204.12.1 14.8 9.9 5.0 150 666 AAIAIIAPAIVP 12 8 50.2 195.8 2.3 14.7 9.9 5.0149 665 LAIVLAAPVAVP 12 8 50.2 203.3 2.3 14.7 9.9 5.0 84 363AVLAVAPALIVP 12 7 50.2 203.3 2.3 14.7 9.8 4.9 57 242 AALLVPALVAAP 12 657.3 187.5 2.1 14.6 9.7 4.9 90 384 VIVAIAPALLAP 12 7 50.2 211.6 2.4 14.09.4 4.7 214 877 VAIIAVPAVVAP 12 7 57.3 195.0 2.4 14.0 9.4 4.7 206 863AAVVLLPIIAAP 12 7 41.3 211.7 2.4 13.8 9.3 4.7 123 525 ALAIVVAPVAVP 12 850.2 195.0 2.4 13.8 9.2 4.7 213 875 AIAIVVPAVAVP 12 7 50.2 195.0 2.413.8 9.2 4.7 69 285 AIVLLPAAVVAP 12 6 50.2 203.3 2.4 13.3 8.9 4.5 65 281ALIVLPAAVAVP 12 6 50.2 203.3 2.4 13.3 8.9 4.5 209 867 ALLVVIAPLAAP 12 841.3 211.7 2.4 13.2 8.8 4.4 172 766 IVVIAVAPAVAP 12 8 50.2 195.0 2.412.9 8.6 4.4 80 342 VIVALAPAVLAP 12 7 50.2 203.3 2.3 12.7 8.5 4.3 217881 AALIVVPAVAVP 12 7 50.2 195.0 2.4 12.7 8.5 4.3 119 505 AIIIVIAPAAAP12 8 50.2 195.8 2.3 12.4 8.3 4.2

TABLE 28 SEQ Proline Rigidity/ Sturctural Relative Ratio ID PositionFlexibility Feature Hydropathy (Fold) NO aMTD Sequences Length (PP) (II)(AI) (GRAVY) A B C 169 763 VAVLIAVPALAP 12 8 57.3 203.3 2.3 12.3 7.2 4.2156 687 AILAVALPLLAP 12 8 57.3 220.0 2.3 12.0 7.0 4.1 159 706IVAVALLPAVAP 12 8 50.2 203.3 2.4 12.0 7.0 4.1 145 643 LALVLAAPAIVP 12 850.2 211.6 2.4 11.8 7.9 4.0 66 282 VLAVAPALIVAP 12 6 50.2 203.3 2.4 11.87.9 4.0 126 543 LLAALIAPAALP 12 8 57.3 204.1 2.1 11.7 7.8 4.0 78 325IVAVALPAVALP 12 7 50.2 203.3 2.3 11.7 7.8 4.0 199 846 IAVAVAAPLLVP 12 841.3 203.3 2.4 11.7 6.8 4.0 89 383 VIVALAPALLAP 12 7 50.2 211.6 2.3 11.67.7 3.9 87 381 VVAIVLPAVAAP 12 7 50.2 195.0 2.4 11.5 7.7 3.9 183 808LVVLAAAPLAVP 12 8 41.3 203.3 2.3 11.5 7.6 3.9 208 865 AVLVIAVPAIAP 12 857.3 203.3 2.5 11.3 7.5 3.8 162 725 IAVLAVAPAVLP 12 8 57.3 203.3 2.311.2 7.5 3.8 197 844 VVALLAPLIAAP 12 7 41.3 211.8 2.4 11.2 7.5 3.8 228897 AVIVPVAIIAAP 12 5 50.2 203.3 2.5 11.2 7.5 3.8 141 605 VIAAVLAPVAVP12 8 57.3 195.0 2.4 11.0 7.4 3.7 166 744 AAVVIVAPVALP 12 8 50.2 195.02.4 11.0 7.3 3.7 51 221 AAILAPIVALAP 12 6 50.2 195.8 2.2 10.9 7.3 3.7142 622 ALIVLAAPVAVP 12 8 50.2 203.3 2.4 10.6 7.1 3.6 92 401AALAVIPAAILP 12 7 54.9 195.8 2.2 10.6 7.1 3.6 77 324 IVAVALPAALVP 12 750.2 203.3 2.3 10.3 6.9 3.5 215 878 IVALVAPAAVVP 12 7 50.2 195.0 2.410.3 6.9 3.5 71 302 LALAPALALLAP 12 5 57.3 204.2 2.1 10.2 6.8 3.4 154685 ALLVAVLPAALP 12 8 57.3 211.7 2.3 10.2 5.9 3.4 201 848 AVAIVVLPAVAP12 8 50.2 195.0 2.4 10.0 6.7 3.4 138 602 VIVALAAPVLAP 12 8 50.2 203.32.4 9.9 5.8 3.4 178 788 AIAVAIAPVALP 12 8 57.3 187.5 2.3 9.8 6.6 3.3 38145 LLAVVPAVALAP 12 6 57.3 203.3 2.3 9.5 6.3 3.2 6 11 VVALAPALAALP 12 657.3 187.5 2.1 9.5 6.3 3.2 35 141 AVIVLPALAVAP 12 6 50.2 203.3 2.4 9.46.3 3.2 120 521 LAALIVVPAVAP 12 8 50.2 203.3 2.4 9.4 6.3 3.2 100 425AVVAIAPVLALP 12 7 57.3 203.3 2.4 9.4 6.3 3.2 86 365 AVIVVAPALLAP 12 750.2 203.3 2.3 9.3 6.2 3.1 62 263 ALAVIPAAAILP 12 6 54.9 195.8 2.2 9.06.0 3.0 82 345 ALLIVAPVAVAP 12 7 50.2 203.3 2.3 8.9 5.9 3.0 203 850LVIALAAPVALP 12 8 57.3 211.7 2.4 8.8 5.9 3.0 37 144 VLAIVPAVALAP 12 650.2 203.3 2.4 8.8 5.9 3.0 173 767 IVVAAVVPALAP 12 8 50.2 195.0 2.4 8.55.0 2.9 47 185 AALVLPLIIAAP 12 6 41.3 220.0 2.4 8.5 5.7 2.9 202 849AVILLAPLIAAP 12 7 57.3 220.0 2.4 8.3 4.8 2.8 40 162 AVVALPAALIVP 12 650.2 203.3 2.4 8.2 5.5 2.8 207 864 ALLVIAPAIAVP 12 7 57.3 211.7 2.4 8.24.8 2.8 42 164 LAAVLPALLAAP 12 6 57.3 195.8 2.1 8.2 5.5 2.8 236 907VAIALAPVVVAP 12 7 57.3 195.0 2.4 8.1 5.4 2.8 103 444 LAAALVPVALVP 12 757.3 203.3 2.3 8.1 5.4 2.7 102 443 ALAALVPVALVP 12 7 57.3 203.3 2.3 8.05.3 2.7 221 887 VLAVAPAVAVLP 12 6 57.3 195.0 2.4 7.7 5.1 2.6 231 901ALVAVLPAVAVP 12 7 57.3 195.0 2.4 7.7 5.1 2.6 167 746 VAIIVVAPALAP 12 850.2 203.3 2.4 7.6 4.4 2.6 232 902 ALVAPLLAVAVP 12 5 41.3 203.3 2.3 7.65.1 2.6 133 565 VAIVLVAPAVAP 12 8 50.2 195.0 2.4 7.5 5.0 2.5 59 245AAALAPVLALVP 12 6 57.3 187.5 2.1 7.5 5.0 2.5 165 743 AIAIALVPVALP 12 857.3 211.6 2.4 7.4 4.9 2.5 109 465 AVVILVPLAAAP 12 7 57.3 203.3 2.4 7.44.9 2.5 30 104 AVVAAPLVLALP 12 6 41.3 203.3 2.3 7.3 4.9 2.5

TABLE 29 SEQ Proline Rigidity/ Sturctural Relative Ratio ID PositionFlexibility Feature Hydropathy (Fold) NO aMTD Sequences Length (PP) (II)(AI) (GRAVY) A B C 160 707 IVALAVLPAVAP 12 8 50.2 203.3 2.4 7.3 4.9 2.5212 872 VLAAAVLPLVVP 12 8 41.3 219.2 2.5 7.3 4.9 2.5 135 583AVILALAPIVAP 12 8 50.2 211.6 2.4 7.3 4.8 2.4 216 879 AAIVLLPAVVVP 12 750.2 219.1 2.5 7.2 4.8 2.4 175 784 VAALPAVALVVP 12 5 57.3 195.0 2.4 7.14.7 2.4 225 893 VIAIPAILAAAP 12 5 54.9 195.8 2.3 7.0 4.7 2.4 8 13AAALVPVVALLP 12 6 57.3 203.3 2.3 7.0 4.7 2.4 184 809 LIVLAAPALAAP 12 750.2 195.8 2.2 7.0 4.7 2.4 104 445 ALAALVPALVVP 12 7 57.3 203.3 2.3 6.94.6 2.3 22 81 AALLPALAALLP 12 5 57.3 204.2 2.1 6.9 4.6 2.3 151 667LAVAIVAPALVP 12 8 50.2 203.3 2.3 6.9 4.6 2.3 235 906 AVIALAPVVVAP 12 757.3 195.0 2.4 6.8 4.6 2.3 112 483 ILAAAIIPAALP 12 8 54.9 204.1 2.2 6.84.5 2.3 114 485 AILAAIVPLAVP 12 8 50.2 211.6 2.4 6.8 4.5 2.3 97 421AAILAAPLIAVP 12 7 57.3 195.8 2.2 6.7 4.5 2.3 136 585 ALIVAIAPALVP 12 850.2 211.6 2.4 6.6 4.4 2.2 99 424 AVVVAAPVLALP 12 7 57.3 195.0 2.4 6.64.4 2.2 85 364 LVAAVAPALIVP 12 7 50.2 203.3 2.3 6.5 4.3 2.2 93 402ALAAVIPAAILP 12 7 54.9 195.8 2.2 6.4 4.3 2.2 106 462 IAAVLVPAVALP 12 757.3 203.3 2.4 6.3 4.2 2.1 64 265 VLAIAPLLAAVP 12 6 41.3 211.6 2.3 6.04.0 2.0 70 301 VIAAPVLAVLAP 12 6 57.3 203.3 2.4 6.0 4.0 2.0 45 183LLAAPVVIALAP 12 6 57.3 211.6 2.4 6.0 4.0 2.0 58 243 AAVLLPVALAAP 12 657.3 187.5 2.1 5.9 3.9 2.0 148 664 ILIAIAIPAAAP 12 8 54.9 204.1 2.3 5.73.8 1.9 174 783 IVALVPAVAIAP 12 6 50.2 203.3 2.5 5.7 3.8 1.9 116 502AIVALAVPVLAP 12 8 50.2 203.3 2.4 5.6 3.7 1.9 61 262 ALIAVPAIIVAP 12 650.2 211.6 2.4 5.5 3.7 1.9 152 683 LAIVLAAPAVLP 12 8 50.2 211.7 2.4 5.53.2 1.9 193 830 IALVAAPVALVP 12 7 57.3 203.3 2.4 5.3 3.5 1.8 170 764AVALAVLPAVVP 12 8 57.3 195.0 2.3 5.0 3.4 1.7 182 807 AVALAVPALVLP 12 757.3 203.3 2.3 5.0 3.3 1.7 46 184 LAAIVPAIIAVP 12 6 50.2 211.6 2.4 4.83.2 1.6 73 305 IALAAPILLAAP 12 6 57.3 204.2 2.2 4.8 3.2 1.6 27 101LVALAPVAAVLP 12 6 57.3 203.3 2.3 4.5 3.0 1.5 72 304 AIILAPIAAIAP 12 657.3 204.2 2.3 4.4 3.0 1.5 140 604 VALIAVAPAVVP 12 8 57.3 195.0 2.4 4.32.5 1.5 146 645 ALAVVALPAIVP 12 8 50.2 203.3 2.4 4.3 2.9 1.5 48 201LALAVPALAALP 12 6 57.3 195.8 2.1 4.2 2.8 1.4 41 163 LALVLPAALAAP 12 657.3 195.8 2.1 4.1 2.4 1.4 195 832 AVAAIVPVIVAP 12 7 43.2 195.0 2.5 4.12.7 1.4 44 182 ALIAPVVALVAP 12 6 57.3 203.3 2.4 4.0 2.7 1.4 11 23VVLVLPAAAAVP 12 6 57.3 195.0 2.4 4.0 2.6 1.3 31 105 LLALAPAALLAP 12 657.3 204.1 2.1 4.0 2.6 1.3 129 561 AAVAIVLPAVVP 12 8 50.2 195.0 2.4 3.92.6 1.3 171 765 AVALAVVPAVLP 12 8 57.3 195.0 2.3 3.8 2.2 1.3 153 684AAIVLALPAVLP 12 8 50.2 211.7 2.4 3.5 2.1 1.2 36 143 AVLAVPAVLVAP 12 657.3 195.0 2.4 3.3 2.2 1.1 118 504 LIVALAVPALAP 12 8 50.2 211.7 2.4 3.32.2 1.1 10 22 AVVLVPVLAAAP 12 6 57.3 195.0 2.4 3.1 2.1 1.1 5 5AAALLPVALVAP 12 6 57.3 187.5 2.1 3.1 2.1 1.0 67 283 AALLAPALIVAP 12 650.2 195.8 2.2 3.1 2.0 1.0 21 65 IAIVAPVVALAP 12 6 50.2 203.3 2.4 3.02.0 1.0 219 883 LAIVPAAIAALP 12 6 50.2 195.8 2.2 3.0 2.0 1.0 33 123AAIIVPAALLAP 12 6 50.2 195.8 2.2 2.9 2.0 1.0

TABLE 30 SEQ Proline Rigidity/ Sturctural Relative Ratio ID PositionFlexibility Feature Hydropathy (Fold) NO aMTD Sequences Length (PP) (II)(AI) (GRAVY) A B C 68 284 ALIAPAVALIVP 12 5 50.2 211.7 2.4 2.8 1.8 0.950 205 ALALVPAIAALP 12 6 57.3 195.8 2.2 2.6 1.7 0.9 14 42 VAALPVVAVVAP12 5 57.3 186.7 2.4 2.5 1.7 0.8 32 121 AIVALPALALAP 12 6 50.2 195.8 2.22.5 1.7 0.8 13 25 IVAVAPALVALP 12 6 50.2 203.3 2.4 2.4 1.6 0.8 12 24IALAAPALIVAP 12 6 50.2 195.8 2.2 2.3 1.6 0.8 49 204 LIAALPAVAALP 12 657.3 195.8 2.2 2.2 1.5 0.8 7 12 LLAAVPAVLLAP 12 6 57.3 211.7 2.3 2.2 1.50.7 15 43 LLAAPLVVAAVP 12 5 41.3 187.5 2.1 2.1 1.4 0.7 29 103ALIAAPILALAP 12 6 57.3 204.2 2.2 2.1 1.4 0.7 23 82 AVVLAPVAAVLP 12 657.3 195.0 2.4 2.1 1.4 0.7 4 4 ALALLPVAALAP 12 6 57.3 195.8 2.1 2.0 1.30.7 26 85 LLVLPAAALAAP 12 5 57.3 195.8 2.1 1.9 1.3 0.7 19 63AALLVPALVAVP 12 6 57.3 203.3 2.3 1.9 1.3 0.7 16 44 ALAVPVALLVAP 12 557.3 203.3 2.3 1.6 1.1 0.5 25 84 AAVAAPLLLALP 12 6 41.3 195.8 2.1 1.51.0 0.5 18 62 VALLAPVALAVP 12 6 57.3 203.3 2.3 1.4 0.9 0.5 24 83LAVAAPLALALP 12 6 41.3 195.8 2.1 1.4 0.9 0.5 28 102 LALAPAALALLP 12 557.3 204.2 2.1 1.4 0.9 0.5 143 623 VAAAIALPAIVP 12 8 50.2 187.5 2.3 0.80.6 0.3 19.6 ± 1.6 13.1 ± 1.1 6.6 ± 0.5

Moreover, compared to reference CPPs (B type: MTM12 and C type: MTD85),novel 240 aMTDs averaged of 13±1.1 (maximum 109.9) and 6.6±0.5 (maximum55.5) fold higher cell-permeability, respectively (Tables 26-31).

TABLE 31 Negative control rP38 MTM12 MTD85 aMTD 19.6 ± 1.6* 13.1 ± 1.1*6.6 ± 0.5* The Average of (Best: 164.2) (Best: 109.9) (Best: 55.5) 240aMTDs *Relative Fold (aMTD in Geo Mean in its comparison to rP38, MTM12or MTD85)

In addition, cell-permeability of 31 rPeptides has been compared withthat of 240 aMTDs (0.3±0.04; Tables 32 and 33).

TABLE 32 Relative SEQ Proline Rigidity/ Sturctural Ratio to ID rPeptidePosition Flexibility Feature Hydropathy aMTD NO ID Sequences Length (PP) (II) (AI) (GRAVY) AVE 907 692 PAPLPPVVILAV 12 1,3,5,6 105.5 186.71.8 0.74 895 26 AAIALAAPLAIV 12 8 18.1 204.2 2.5 0.65 891 113PVAVALLIAVPP 12 1,11,12 57.3 195.0 2.1 0.61 897 466 IIAAAAPLAIIP 12 7,1222.8 204.2 2.3 0.52 898 167 VAIAIPAALAIP 12 6,12 20.4 195.8 2.3 0.50 93697 ALLAAPPALLAL 12 6,7 57.3 204.2 2.1 0.41 909 390 VPLLVPVVPVVP 122,6,9,12 105.4 210.0 2.2 0.41 900 426 AAALAIPLAIIP 12 7,12 4.37 204.22.2 0.40 937 214 ALIVAPALMALP 12 6,12 60.5 187.5 2.2 0.33 913 68VAPVLPAAPLVP 12 3,6,9,12 105.5 162.5 1.6 0.32 941 39 CYNTSPCTGCCY 12 652.5 0.0 0.0 0.29 888 934 LILAPAAVVAAA 12 5 57.3 195.8 2.5 0.28 916 938VPVLLPVVVPVP 12 2,6,10,12 121.5 210.0 2.2 0.28 917 329 LPVLVPVVPVVP 122,6,9,12 121.5 210.0 2.2 0.23 901 606 AAAIAAIPIIIP 12 8,12 4.4 204.2 2.40.20 918 49 VVPAAPAVPVVP 12 3,6,9,12 121.5 145.8 1.7 0.18 944 139TGSTNSPTCTST 12 7 53.4 0.0 -0.7 0.17 919 772 LPVAPVIPIIVP 12 2,5,8,1279.9 210.8 2.1 0.16 931 921 IWWFVVLPLVVP 12 8,12 41.3 194.2 2.2 0.14 90266 AGVLGGPIMGVP 12 7,12 35.5 121.7 1.3 0.13 922 693 AAPVLPVAVPIV 123,6,10 82.3 186.7 2.1 0.13 945 18 NYCCTPTTNGQS 12 6 47.9 0.0 -0.9 0.10890 16 NNSCTTYTNGSQ 12 None 47.4 0.0 -1.4 0.08 904 227 LAAIVPIAAAVP 126,12 34.2 187.5 2.2 0.08 905 17 GGCSAPQTTCSN 12 6 51.6 8.3 -0.5 0.08 90667 LDAEVPLADDVP 12 6,12 34.2 130 0.3 0.08 947 635 GSTGGSQQNNQY 12 None31.9 0.0 -1.9 0.07 924 29 VLPPLPVLPVLP 12 3,4,6,9,12 121.5 202.5 1.70.07 949 57 QNNCNTSSQGGG 12 None 52.4 0.0 -1.6 0.06 951 700 GTSNTCQSNQNS12 None 19.1 0.0 -1.6 0.05 952 38 YYNQSTCGGQCY 12 ND 53.8 0.0 -1.0 0.05AVE 0.3 ±0.04

TABLE 33 Relative Ratio to aMTD AVE* rPeptide 0.3 ± 0.04 The Average of31 aMTDs *Out of 240 aMTDs, average relative fold of aMTD had been 19.6fold compared to type A (rP38).

In summary, relative cell-permeability of aMTDs has shown maximum of164.0, 109.9 and 55.5 fold higher to rP38, MTM12 and MTD85,respectively. In average of total 240 aMTD sequences, 19.6±1.6, 13.1±1.1and 6.6±0.5 fold higher cell-permeability are shown to the rP38, MTM12and MTD85, respectively (Tables 26-31). Relative cell-permeability ofnegative control (rP38) to the 240 aMTDs is only 0.3±0.04 fold.

4-5. Intracellular Delivery and Localization of aMTD-Fused RecombinantProteins

Recombinant proteins fused to the aMTDs were tested to determine theirintracellular delivery and localization by laser scanning confocalmicroscopy with a negative control (rP38) and previous published CPPs(MTM12 and MTD85) as the positive control references. NIH3T3 cells wereexposed to 10 μM of FITC-labeled protein for 1 hour at 37, and nucleiwere counterstained with DAPI. Then, cells were examined by confocallaser scanning microscopy (FIG. 7). Recombinant proteins fused to aMTDsclearly display intracellular delivery and cytoplasmic localization(FIG. 7) that are typically higher than the reference CPPs (MTM12 andMTD85). The rP38-fused recombinant protein did not show internalizedfluorescence signal (FIG. 7a ). In addition, as seen in FIG. 8,rPeptides (his-tagged CRA recombinant proteins fused to each rPeptide)display lower- or non-cell-permeability.

4-6. Summary of Quantitative and Visual Cell-Permeability of NewlyDeveloped aMTDs

Histidine-tagged aMTD-fused cargo recombinant proteins have been greatlyenhanced in their solubility and yield. Thus, FITC-conjugatedrecombinant proteins have also been tested to quantitate and visualizeintracellular localization of the proteins and demonstrated highercell-permeability compared to the reference CPPs.

In the previous studies using the hydrophobic signal-sequence-derivedCPPs—MTS/MTM or MTDs, 17 published sequences have been identified andanalyzed in various characteristics such as length, molecular weight, p1value, bending potential, rigidity, flexibility, structural feature,hydropathy, amino acid residue and composition, and secondary structureof the peptides. Based on these analytical data of the sequences, novelartificial and non-natural peptide sequences designated as advanced MTDs(aMTDs) have been invented and determined their functional activity inintracellular delivery potential with aMTD-fused recombinant proteins.

aMTD-fused recombinant proteins have promoted the ability of proteintransduction into the cells compared to the recombinant proteinscontaining rPeptides and/or reference hydrophobic CPPs (MTM12 andMTD85). According to the results, it has been demonstrated that criticalfactors of cell-penetrating peptide sequences play a major role todetermine peptide-mediated intracellular delivery by penetrating plasmamembrane. In addition, cell-permeability can considerably be improved byfollowing the rational that all satisfy the critical factors.

5. Structure/Sequence Activity Relationship (SAR) of aMTDs on DeliveryPotential

After determining the cell-permeability of novel aMTDs,structure/sequence activity relationship (SAR) has been analyzed foreach critical factor in selected some of and all of novel aMTDs (FIGS.13 to 16 and Table 34).

TABLE 34 Rank of Rigidity/ Sturctural Relative Ratio Amino Acid DeliveryFlexibility Feature Hydropathy (Fold) Composition Potential (II) (Al)(GRAVY) A B C A V I L  1~10 55.9 199.2 2.3 112.7 75.5 38.1 4.0 3.5 0.42.1 11~20 51.2 205.8 2.4 56.2 37.6 19.0 4.0 2.7 1.7 1.6 21~30 49.1 199.22.3 43.6 28.9 14.6 4.3 2.7 1.4 1.6 31~40 52.7 201.0 2.4 34.8 23.3 11.84.2 2.7 1.5 1.6 41~50 53.8 201.9 2.3 30.0 20.0 10.1 4.3 2.3 1.1 2.351~60 51.5 205.2 2.4 23.5 15.7 7.9 4.4 2.1 1.5 2.0 222~231 52.2 197.22.3 2.2 1.5 0.8 4.5 2.1 1.0 2.4 232~241 54.1 199.7 2.2 1.7 1.2 0.6 4.61.7 0.2 3.5

5-1. Proline Position:

In regards to the bending potential (proline position: PP), aMTDs withits proline at 7′ or 8′ amino acid in their sequences have much highercell-permeability compared to the sequences in which their prolineposition is at 5′ or 6′ (FIGS. 14a and 15a ).

5-2. Hydropathy:

In addition, when the aMTDs have GRAVY (Grand Average of Hydropathy)ranging in 2.1-2.2, these sequences display relatively lowercell-permeability, while the aMTDs with 2.3-2.6 GRAVY are shownsignificantly higher one (FIGS. 14b and 15b ).

5-3. rPeptide SAR:

To the SAR of aMTDs, rPeptides have shown similar SAR correlations inthe cell-permeability, pertaining to their proline position (PP) andhydropathy (GRAVY). These results confirms that rPeptides with highGRAVY (2.4 [H]-2.6) have better cell-permeability (FIG. 16).

5-4. Analysis of Amino Acid Composition:

In addition to proline position and hydropathy, the difference of aminoacid composition is also analyzed. Since aMTDs are designed based oncritical factors, each aMTD-fused recombinant protein has equally twoproline sequences in the composition. Other hydrophobic and aliphaticamino acids—alanine, isoleucine, leucine and valine —are combined toform the rest of aMTD peptide sequences.

Alanine: In the composition of amino acids, the result does not show asignificant difference by the number of alanine in terms of the aMTD'sdelivery potential because all of the aMTDs have three to five alanines.In the sequences, however, four alanine compositions show the mosteffective delivery potential (geometric mean) (FIG. 13a ).

Leucine and Isoleucine: Also, the compositions of isoleucine and leucinein the aMTD sequences show inverse relationship between the number ofamino acid (I and L) and delivery potential of aMTDs. Lower number ofisoleucine and leucine in the sequences tends to have higher deliverypotential (geometric mean) (FIGS. 13b and 13c ).

Valine: Conversely, the composition of valine of aMTD sequences showspositive correlation with their cell-permeability. When the number ofvaline in the sequence is low, the delivery potential of aMTD is alsorelatively low (FIG. 13d ).

Ten aMTDs having the highest cell-permeability are selected (averagegeometric mean: 2584±126). Their average number of valine in thesequences is 3.5; 10 aMTDs having relatively low cell-permeability(average geometric mean: 80±4) had average of 1.9 valine amino acids.The average number of valine in the sequences is lowered as theircell-permeability is also lowered as shown in FIG. 13d . Compared tohigher cell-permeable aMTDs group, lower sequences had average of 1.9 intheir valine composition. Therefore, to obtain high cell-permeablesequence, an average of 2-4 valines should be composed in the sequence.

5-5. Conclusion of SAR Analysis:

As seen in FIG. 15, all 240 aMTDs have been examined for theseassociation of the cell-permeability and the critical factors: bendingpotential (PP), rigidity/flexibility (II), structure feature (AI), andhydropathy (GRAVY), amino acid length and composition. Through thisanalysis, cell-permeability of aMTDs tends to be lower when theircentral proline position is at 5′ or 6′ and GRAVY is 2.1 or lower (FIG.15). Moreover, after investigating 10 higher and 10 lower cell-permeableaMTDs, these trends are clearly shown to confirm the association ofcell-permeability with the central proline position and hydropathy.

6. Experimental Confirmation of Index Range/Feature of Critical Factors

The range and feature of five out of six critical factors have beenempirically and experimentally determined that are also included in theindex range and feature of the critical factors initially proposedbefore conducting the experiments and SAR analysis. In terms of indexrange and feature of critical factors of newly developed 240 aMTDs, thebending potential (proline position: PP), rigidity/flexibility(Instability Index: II), structural feature (Aliphatic Index: AI),hydropathy (GRAVY), amino acid length and composition are all within thecharacteristics of the critical factors derived from analysis ofreference hydrophobic CPPs.

Therefore, our hypothesis to design and develop new hydrophobic CPPsequences as advanced MTDs is empirically and experimentally proved anddemonstrated that critical factor-based new aMTD rational design iscorrect.

TABLE 35 Summarized Critical Factors of aMTD Analysis of Newly DesignedExperimental CPPs Results Critical Factor Range Range Bending PotentialProline presences Proline presences (Proline Position: PP) in the middlein the middle (5′, 6′, 7′ (5′, 6′, 7′ or 8′) and at the or 8′) and atthe end of peptides end of peptides Rigidity/Flexibility 40-60 41.3-57.3(Instability Index: II) Structural Feature 180-220 187.5-220.0(Aliphatic Index: AI) Hydropathy 2.1-2.6 2.2-2.6 (Grand Average ofHydropathy GRAVY) Length  9-13 12 (Number of Amino Acid) Amino acidComposition A, V, I, L, P A, V, I, L, P

7. Discovery and Development of Protein-Based New Biotherapeutics withMITT Enabled by aMTDs for Protein Therapy

Total of 240 aMTD sequences have been designed and developed based onthe critical factors. Quantitative and visual cell-permeability of 240aMTDs (hydrophobic, flexible, bending, aliphatic and 12 a/a-lengthpeptides) are all practically determined.

To measure the cell-permeability of aMTDs, rPeptides have also beendesigned and tested. As seen in FIGS. 13 to 15, there are vividassociation of cell-permeability and the critical factors of thepeptides. Out of these critical factors, we are able to configure thatthe most effective cell-permeable aMTDs have the amino acid length of12; composition of A, V, L, I and P; multiple proline located at either7′ or 8′ and at the end (12′); instability index ranged of 41.3-57.3;aliphatic index ranged of 187.5-220.0; and hydropathy (GRAVY) ranged of2.2-2.6.

These examined critical factors are within the range that we have setfor our critical factors; therefore, we are able to confirm that theaMTDs that satisfy these critical factors have relatively highcell-permeability and much higher intracellular delivery potentialcompared to reference hydrophobic CPPs reported during the past twodecades.

It has been widely evident that many human diseases are caused byproteins with deficiency or over-expression that causes mutations suchas gain-of-function or loss-of-function. If biologically active proteinscould be delivered for replacing abnormal proteins within a short timeframe, possibly within an hour or two, in a quantitative manner, thedosage may be regulated depending on when and how proteins may beneeded. By significantly improving the solubility and yield of novelaMTD in this invention (Table 31), one could expect its practicalpotential as an agent to effectively deliver therapeutic macromoleculessuch as proteins, peptides, nucleic acids, and other chemical compoundsinto live cells as well as live mammals including human. Therefore,newly developed MITT utilizing the pool (240) of novel aMTDs can be usedas a platform technology for discovery and development of protein-basedbiotherapeutics to apprehend intracellular protein therapy afterdetermining the optimal cargo-aMTD relationship.

The following examples are presented to aid practitioners of theinvention, to provide experimental support for the invention, and toprovide model protocols. In no way are these examples to be understoodto limit the invention.

Example 1. Development of Novel Advanced Macromolecule TransductionDomain (aMTD)

H-regions of signal sequences (HRSP)-derived CPPs (MTS/MTM and MTD) donot have a common sequence, a sequence motif, and/or a common structuralhomologous feature. In this invention, the aim is to develop improvedhydrophobic CPPs formatted in the common sequence and structural motifthat satisfy newly determined ‘critical factors’ to have a ‘commonfunction,’ to facilitate protein translocation across the plasmamembrane with similar mechanism to the analyzed CPPs.

The structural motif as follows:

Here, X(s) independently refer to Alanine (A), Valine (V), Leucine (L)or Isoleucine (I); Proline (P) can be positioned in one of U(s) (either5′, 6′, 7′ or 8′) and the remaining U(s) are independently composed ofA, V, L or I; and P at the 12′ is Proline.

In Table 9, universal common sequence/structural motif is provided asfollows. The amino acid length of the peptides in this invention rangesfrom 9 to 13 amino acids, mostly 12 amino acids, and their bendingpotentials are dependent with the presence and location of proline inthe middle of sequence (at 5′, 6′, 7′ or 8′ amino acid) and at the endof peptide (at 12′) for recombinant protein bending. Instability index(II) for rigidity/flexibility of aMTDs is II<40, grand average ofhydropathy (GRAVY) for hydropathy is around 2.2, and aliphatic index(AI) for structural features is around 200 (Table 9). Based on thesestandardized critical factors, new hydrophobic peptide sequences, namelyadvanced macromolecule transduction domain peptides (aMTDs), in thisinvention have been developed and summarized in Tables 10 to 15.

Example 2. Construction of Expression Vectors for Recombinant ProteinsFused to aMTDs

Our newly developed technology has enabled us to expand the method formaking cell-permeable recombinant proteins. The expression vectors weredesigned for histidine-tagged CRA proteins fused with aMTDs orrPeptides. To construct expression vectors for recombinant proteins,polymerase chain reaction (PCR) had been devised to amplify eachdesigned aMTD or rPeptide fused to CRA.

The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mMdNTP mixture, 1× reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctorprotein, Korea) was digested on the restriction enzyme site between NdeI (5′) and Sal I (3′) involving 35 cycles of denaturation (95° C.),annealing (62° C.), and extension (72° C.) for 30 seconds each. For thelast extension cycle, the PCR reactions remained for 5 minutes at 72° C.Then, they were cloned into the site of pET-28a(+) vectors (Novagen,Darmstadt, Germany). DNA ligation was performed using T4 DNA ligase at4° C. overnight. These plasmids were mixed with competent cells of E.coli DH5-alpha strain on the ice for 10 minutes. This mixture was placedon the ice for 2 minutes after it was heat shocked in the water bath at42° C. for 90 seconds. Then, the mixture added with LB broth media wasrecovered in 37° C. shaking incubator for 1 hour. Transformant wasplated on LB broth agar plate with kanamycin (50 μg/mL) (Biopure,Johnson City, Tenn., USA) before incubating at 37° C. overnight. From asingle colony, plasmid DNA was extracted, and after the digestion of NdeI and Sal I restriction enzymes, digested DNA was confirmed at 645 bp byusing 1.2% agarose gels electrophoresis (FIG. 2).

Example 3. Inducible Expression, Purification and Preparation ofRecombinant Proteins Fused to aMTDs and rPeptides

To express recombinant proteins, pET-28a(+) vectors for the expressionof CRA proteins fused to a negative control [rPeptide 38 (rP38)],reference hydrophobic CPPs (MTM₁₂ and MTD85) and aMTDs were transformedin E. coli BL21 (DE3) strains. Cells were grown at 37° C. in LB mediumcontaining kanamycin (50 μg/ml) with a vigorous shaking and induced atOD₆₀₀=0.6 by adding 0.7 mM IPTG (Biopure) for 2 hours at 37° C. Inducedrecombinant proteins were loaded on 15% SDS-PAGE gel and stained withCoomassie Brilliant Blue (InstantBlue, Expedeon, Novexin, UK) (FIG. 3).

The E. coli cultures were harvested by centrifugation at 5,000×rpm for10 minutes, and the supernatant was discarded. The pellet wasre-suspended in the lysis buffer (50 mM NaH₂PO₄, 10 mM Imidazol, 300 mMNaCl, pH 8.0). The cell lysates were sonicated on ice using a sonicator(Sonics and Materials, Inc., Newtown, Conn., USA) equipped with a probe.After centrifuging the cell lysates at 5,000×rpm for 10 minutes topellet the cellular debris, the supernatant was incubated with lysisbuffer-equilibrated Ni-NTA resin (Qiagen, Hilden, Germany) gently byopen-column system (Bio-rad, Hercules, Calif., USA). After washingprotein-bound resin with 200 ml wash buffer (50 mM NaH₂PO₄, 20 mMImidazol, 300 mM NaCl, pH 8.0), the bounded proteins were eluted withelution buffer (50 mM NaH₂PO₄, 250 mM Imidazol, 300 mM NaCl, pH 8.0).

Recombinant proteins purified under natural condition were analyzed on15% SDS-PAGE gel and stained with Coomassie Brilliant Blue (FIG. 4). Allof the recombinant proteins were dialyzed for 8 hours and overnightagainst physiological buffer, a 1:1 mixture of cell culture medium(Dulbecco's Modified Eagle's Medium: DMEM, Hyclone, Logan, Utah, USA)and Dulbecco's phosphate buffered saline (DPBS, Gibco, Grand Island,N.Y., USA). From 316 aMTDs and 141 rPeptides cloned, 240 aMTD- and 31rPeptide-fused recombinant proteins were induced, purified, prepared andanalyzed for their cell-permeability.

Example 4. Determination of Quantitative Cell-Permeability ofRecombinant Proteins

For quantitative cell-permeability, the aMTD- or rPeptide-fusedrecombinant proteins were conjugated to fluorescein isothiocyanate(FITC) according to the manufacturer's instructions (Sigma-Aldrich, St.Louis, Mo., USA). RAW 264.7 cells were treated with 10 μM FITC-labeledrecombinant proteins for 1 hour at 37° C., washed three times with coldPBS, treated with 0.25% tripsin/EDTA (Sigma-Aldrich, St. Louis, Mo.) for20 minutes at 37° C. to remove cell-surface bound proteins.Cell-permeability of these recombinant proteins were analyzed by flowcytometry (Guava, Millipore, Darmstadt, Germany) using the FlowJocytometric analysis software (FIGS. 5 to 6). The relativecell-permeability of aMTDs were measured and compared with the negativecontrol (rP38) and reference hydrophobic CPPs (MTM12 and MTD85) (Table31).

Example 5. Determination of Cell-Permeability and IntracellularLocalization of Recombinant Proteins

For a visual reference of cell-permeability, NIH3T3 cells were culturedfor 24 hours on coverslip in 24-wells chamber slides, treated with 10 μMFITC-conjugated recombinant proteins for 1 hour at 37° C., and washedthree times with cold PBS. Treated cells were fixed in 4%paraformaldehyde (PFA, Junsei, Tokyo, Japan) for 10 minutes at roomtemperature, washed three times with PBS, and mounted with VECTASHIELDMounting Medium (Vector laboratories, Burlingame, Calif., USA), andcounter stained with DAPI (4′,6-diamidino-2-phenylindole). Theintracellular localization of the fluorescent signal was determined byconfocal laser scanning microscopy (LSM700, Zeiss, Germany; FIGS. 7athrough 7k , and 8).

Example 6-1. Cloning of aMTD/SD-fused SOCS3 recombinant proteinFull-length cDNA for human SOCS3 (SEQ ID NO: 815) was purchased fromOrigene (USA). Histidine-tagged human SOCS3 proteins were constructed byamplifying the SOCS3 cDNA (225 amino acids) using primers for aMTD fusedto SOCS3 cargo. The PCR reactions (100 ng genomic DNA, 10 pmol eachprimer, each 0.2 mM dNTP mixture, lx reaction buffer and 2.5 U Pfu(+)DNA polymerase (Doctor protein, Korea)) were digested on the restrictionenzyme site between Nde I (5′) and Sal I (3′) involving 35 cycles ofdenaturing (95° C.), annealing (62° C.), and extending (72° C.) for 45sec each. For the last extension cycle, the PCR reactions remained for10 min at 72° C. The PCR products were subcloned into 6×His expressionvector, pET-28a(+) (Novagen, Darmstadt, Germany). Coding sequence forSDA or SDB fused to C terminus of his-tagged aMTD-SOCS3 was cloned atBamHI (5′) and Sal1 (3′) in pET-28a(+) from PCR-amplified DNA segmentsand confirmed by DNA sequence analysis of the resulting plasmids.

TABLE 36 Recombinant Cargo SD Protein 5′-primers 3′-pimers SOCS3 — HS35′-GGAATTCCATATGGTCACCC 5′-CCCGGATCCTTAAAGCGGGGC ACAGCAAGTTTCCCGCCGCC-3′ATCGTACTGGTCCAGGAA-3′ (SEQ ID NO: 955) (SEQ ID NO: 956) — HM₁₆₅S35′-GGAATTCCATATGGCGCTGG 5′-CCCGGATCCTTAAAGCGGGGC CGGTGCCGGTGGCGCTGGCGATATCGTACTGGTCCAGGAA-3′ TGTGCCGGTCACCCACAGCAAG (SEQ ID NO: 958) TTTC-3′(SEQ ID NO: 957) A HM₁₆₅S3A 5′-GGAATTCCATATGGCGCTGG5′-CGCGTCGACTTACCTCGGCTG CGGTGCCGGTGGCGCTGGCGAT CACCGGCACGGCGATAC-3′TGTGCCGGTCACCCACAGCAAG (SEQ ID NO: 960) TTTC-3′ (SEQ ID NO: 959) BHM₁₆₅S3B 5′-GGAATTCCATATGGCGCTGG 5′-CGCGTCGACTTAAAGGGTTTCCGGTGCCGGTGGCGCTGGCGAT CGAAGGCTTGGCTATCTT-3′ TGTGCCGGTCACCCACAGCAAG(SEQ ID NO: 962) TTTC-3′ (SEQ ID NO: 961) C HM₁₆₅S3C5′-GGAATTCCATATGGCGCTGG 5′-GCGTCGACTTAGGCCAGGTTA CGGTGCCGGTGGCGCTGGCGATGCGTCGAG-3′ TGTGCCGGTCACCCACAGCAAG (SEQ ID NO: 964) TTTC-3′(SEQ ID NO: 963) D HM₁₆₅S3D 5′-GGAATTCCATATGGCGCTGG5′-GCGTCGACTTATTTTTTCTCGG CGGTGCCGGTGGCGCTGGCGAT ACAGATA-3′TGTGCCGGTCACCCACAGCAAG (SEQ ID NO: 966) TTTC-3′ (SEQ ID NO: 965) EHM₁₆₅S3E 5′-GGAATTCCATATGGCGCTGG 5′-ACGCGTCGACTTAACCTCCAACGGTGCCGGTGGCGCTGGCGAT TCTGTTCGCGGTGAGCCTC-3′ TGTGCCGGTCACCCACAGCAAG(SEQ ID NO: 968) TTTC-3′ (SEQ ID NO: 967)

Example 6-2. Preparation of aMTD/SD-Fused SOCS3 Recombinant Protein

To determine a stable structure of the cell-permeable aMTD/SD-fusedSOCS3 recombinant protein, a pET-28a(+) vector and an E. coliBL21-CodonPlus (DE3)-RIL were subjected to the following experiment.

Each of the recombinant expression vectors, HS3, HMS3, HMS3A, HMS3B,HMS3C, HMS3D, and HMS3E prepared in example 6-1 was transformed into E.coli BL21 CodonPlus(DE3)-RIL by a heat shock method, and then 600 ul ofeach was incubated in an LB medium (Biopure, Johnson City, Tenn., USA)containing 50 μg/ml, of kanamycin at 37° C. for 1 hour. Thereafter, therecombinant protein gene-introduced E. coli was inoculated in 7 ml of LBmedium, and then incubated at 37° C. overnight. The E. coli wasinoculated in 700 ml of LB medium and incubated at 37° C. until OD₆₀₀reached 0.6. To this culture medium, 0.6 mM ofisopropyl-β-D-thiogalactoside (IPTG, Gen Depot, USA) was added as aprotein expression inducer, followed by further incubation at 37° C. for3 hours. This culture medium was centrifuged at 4° C. and 8,000 rpm for10 minutes and a supernatant was discarded to recover a cell pellet. Thecell pellet thus recovered was suspended in a lysis buffer (100 mMNaH₂PO₄, 10 mM Tris-HCl, 8 M Urea, pH 8.0), and cells were disrupted bysonication (on/off time: 30 sec/30 sec, on time 2 hours, amplify 40%),and centrifuged at 15,000 rpm for 30 min to obtain a soluble fractionand an insoluble fraction.

This insoluble fraction was suspended in a denature lysis buffer (8 MUrea, 10 mM Tris, 100 mM Sodium phosphate) and purified by Ni²⁺ affinitychromatography as directed by the supplier(Qiagen, Hilden, Germany) andrefolded by dialyzing with a refolding buffer (0.55 M guanidine HCl,0.44 M L-arginine, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 100 mM NDSB,2 mM reduced glutathione, and 0.2 mM oxidized glutathione). Afterpurification, the proteins were put in a SnakeSkin Dialysis Tubing bag(pore size: 10000 mw, Thermo scientific, USA) and then they weredialyzed by physiological buffer (DMEM). The strain lysate where proteinexpression was not induced, the strain lysate where protein expressionwas induced by addition of IPTG, and purified proteins were loaded onSDS-PAGE to analyze protein expression characteristics and expressionlevels (FIGS. 19 and 20).

As shown in FIG. 19, it was confirmed that SOCS3 recombinant proteinsshowed high expression levels in the BL21CodonPlus(DE3)-RIL strain.SOCS3 recombinant proteins containing aMTD₁₆₅ and solubilization domain(HM₁₆₅S3A and HM₁₆₅S3B) had little tendency to precipitate whereasrecombinant SOCS3 proteins lacking a solubilization domain (HM₁₆₅S3) orlacking an aMTD and a SD (HS3) were largely insoluble. Solubility ofaMTD/SD-fused SOCS3 proteins was scored on a 5 point scale compared withthat of SOCS3 proteins lacking the solubilization domain.

Example 6-3. Determination of Solubility/Yield of aMTD/SD-Fused SOCS3Recombinant Proteins According to SD Type

To determine aMTD/SD-fused SOCS3 recombinant proteins having optimalcell-permeability, solubilization domains were replaced in the samemanner as in Example 6-2 to prepare 5 kinds of aMTD/SD-fused SOCS3recombinant proteins, and their solubility/yield were measured (FIG.19).

As shown in FIG. 19, it was confirmed that the aMTD/SD-fused SOCS3recombinant protein prepared by fusing with SDB among the different SDsshowed the highest solubility/yield. Therefore, the SDB-fused iCP-SOCS3recombinant protein was used in the subsequent experiment.

Example 6-4. Comparison Between aMTD/SD-Fused SOCS3 Recombinant Proteinand Cationic CPP/SD-Fused SOCS3 Recombinant Protein

To compare the solubility/yield, cell/tissue-permeability, mechanism ofcytopermeability of aMTD/SD-fused SOCS3 recombinant proteins to those ofconventional cationic CPP/SD-fused SOCS3 recombinant proteins, cloning,preparation, and measurement of solubility/yield of the cationicCPP/SD-fused SOCS3 recombinant proteins were performed in the samemanner as in Examples 6-1 to 6-3 except for a known cationic CPP (TAT orPolyR) being used instead of aMTD. Sequences of amino acids andnucleotides of cationic CPP, and the primers used in this example areshown in FIG. 71.

The solubility/yield of aMTD165/SD-fused SOCS3 recombinant proteins wasmuch higher than that of TAT/SD-fused SOCS3 or PolyR/SD-fused SOCS3recombinant proteins (FIG. 72b ).

Example 7-1. Cell-Permeability Test

To examine cell-permeability of SOCS3 recombinant protein, SOCS3recombinant proteins were conjugated to 5/6-fluorescein isothiocyanate(FITC). RAW 264.7 (KCLB, Seoul, South Korea) (FIG. 20) or NIH3T3 cells(KCLB, Seoul, South Korea) (FIG. 21) were treated with 10 μMFITC-labeled SOCS3 recombinant proteins and cultivated for 1 hr at 37°C.

In this regard, RAW 264.7 cells were cultured in a DMEM mediumcontaining 10% fetal bovine serum (FBS, Hyclone, USA) and 500 mg/ml of1% penicillin/streptomycin (Hyclone, USA).

After cultivation, the cells were washed three times with ice-cold PBS(Phosphate-buffered saline, Hyclone, USA) and treated with proteinase K(10 μg/mL, SIGMA, USA) to remove surface-bound proteins, andinternalized proteins were measured by flow cytometry (FlowJo cytometricanalysis software, Guava, Millipore, Darmstadt, Germany). Untreatedcells (gray) and equimolar concentration of unconjugated FITC (FITConly, green)-treated cells were served as control (FIG. 20). Each ofNIH3T3 cells was incubated for 1 hour at 37° C. with 10 μM FITC-labeledSOCS3 protein. For nuclear staining, a mixture of VECTASHIELD MountingMedium (Vector laboratories, Burlingame, Calif.) and DAPI(4′,6-diamidino-2-phenylindole) was added to NIH3T3 cells, andvisualized using a confocal laser microscope (LSM700, Zeiss, Germany)(FIG. 21).

As shown in FIGS. 20 and 21, SOCS3 recombinant proteins containingaMTD₁₆₅ (HM₁₆₅S3, HM₁₆₅S3A and HM₁₆₅S3B) efficiently entered the cells(FIGS. 20 and 21) and were localized to various extents in cytoplasm(FIG. 21). In contrast, SOCS3 protein containing non-aMTD (HS3) did notappear to enter cells. While all SOCS3 proteins containing aMTD₁₆₅transduced into the cells, HM₁₆₅S3B displayed more uniform cellulardistribution, and protein uptake of HM₁₆₅S3B was also very efficient.

Example 7-1-2. Comparison Between aMTD/SD-Fused SOCS3 RecombinantProtein and Cationic CPP/SD-Fused SOCS3 Recombinant Protein

The cell-permeability of cationic CPP/SD-fused SOCS3 recombinantproteins was assessed by the same method as used in Example 7-1 exceptfor a known cationic CPP (TAT or PolyR) being used instead of aMTD. Theresults of the assessment were shown in FIGS. 73a and 73 b.

According to the results, all recombinant proteins exhibitedcell-permeability. Among the proteins, aMTD/SD-fused SOCS3 recombinantprotein (HM₁₆₅S3B) showed the highest cell-permeability.

Example 7-2. Tissue-Permeability Test

To further investigate in vivo delivery of SOCS3 recombinant proteins,ICR mice (Doo-Yeol Biotech Co. Ltd., Seoul, Korea) were intraperitoneal(IP) injected with 600 μg/head of 10 μM FITC (Fluoresceinisothiocyanate, SIGMA, USA)-labeled SOCS3 proteins and sacrificed after2 hrs. From the mice, the liver, kidney, spleen, lung, heart, and brainwere removed and washed with PBS, and then placed on a dry ice, andembedded with an O.C.T. compound (Sakura). After cryosectioning at 20μm, tissue distributions of fluorescence-labeled-SOCS3 proteins indifferent organs was analyzed by fluorescence microscopy (Carl Zeiss,Gottingen, Germany)(FIG. 22).

As shown in FIG. 22, SOCS3 recombinant proteins fused to aMTD₁₆₅(HM₁₆₅S3, HM₁₆₅S3A and HM₁₆₅S3B) were distributed to a variety oftissues (liver, kidney, spleen, lung, heart and, to a lesser extent,brain). Liver showed highest levels of fluorescent cell-permeable SOCS3since intraperitoneal administration favors the delivery of proteins tothis organ via the portal circulation. SOCS3 containing aMTD₁₆₅ wasdetectable to a lesser degree in lung, spleen and heart. aMTD/SDB-fusedSOCS3 recombinant protein (HM₁₆₅S3B) showed the highest systemicdelivery of SOCS3 protein to the tissues compared to the SOCS3containing only aMTD (HM₁₆₅S3) or aMTD/SDA (HM₁₆₅S3A) proteins. Thesedata suggest that SOCS3 protein containing both of aMTD₁₆₅ and SDB leadsto higher cell-/tissue-permeability due to the increase in solubilityand stability of the protein, and it displayed a dramatic synergiceffect on cell-/tissue-permeability.

Example 7-2-2. Comparison Between aMTD/SD-Fused SOCS3 RecombinantProtein and Basic CPP/SD-Fused SOCS3 Recombinant Protein

The tissue-permeability of cationic CPP/SD-fused SOCS3 recombinantproteins was assessed by the same method as used in Example 7-2 exceptfor a known cationic CPP (TAT or PolyR) being used instead of aMTD. Theresults of the assessment were shown in FIG. 74.

According to the results, only aMTD/SD-fused SOCS3 recombinant protein(HM₁₆₅S3B) exhibited superior cell-permeability.

Example 8-1 Biological Activity Test of iCP-SOCS3-Inhibition Activity ofIFN-γ-Induced STAT Phosphorylation

It was examined whether the iCP-SOCS3 recombinant proteins prepared byfusion with combinations of aMTD and SD inhibits activation of theJAK/STAT-signaling pathway.

PANC-1 Cells (KCLB, Seoul, South Korea) were treated with 10 ng/ml IFN-γ(R&D systems, Abingdon, UK) for 15 min and treated with either DMEM(vehicle) or 10 μM aMTD/SD-fused SOCS3 recombinant proteins for 2 hrs.The cells were lysed in RIPA lysis buffer (Biosesang, Seongnam, Korea)containing proteinase inhibitor cocktail (Roche, Indianapolis, Ind.,USA), incubated for 15 min at 4° C., and centrifuged at 13,000 rpm for10 min at 4° C. Equal amounts of lysates were separated on 10% SDS-PAGEgels and transferred to a nitrocellulose membrane. The membranes wereblocked using 5% skim milk in TBST and for western blot analysisincubated with the following antibodies: anti-phospho-STAT1 (CellSignaling Technology, USA) and anti-phospho-STAT3 (Cell SignalingTechnology, USA), then HRP conjugated anti-rabbit secondary antibody(Santacruz).

The well-known cytokine signaling inhibitory actions of SOCS3 areinflammation inhibition through i) inhibition of IFN-γ-mediated JAK/STATand ii) inhibition of LPS-mediated cytokine secretion. The ultimate testof cell-penetrating efficiency is a determination of intracellularactivity of SOCS3 proteins transported by aMTD. Since endogenous SOCS3are known to block phosphorylation of STAT1 and STAT3 by IFN-γ-mediatedJanus kinases (JAK) 1 and 2 activation, we demonstrated whethercell-permeable SOCS3 inhibits the phosphorylation of STATs. As shown inFIG. 23, All SOCS3 recombinant proteins containing aMTD (HM₁₆₅S3,HM₁₆₅S3A and HM₁₆₅S3B), suppressed IFN-γ-induced phosphorylation ofSTAT1 and STAT3. In contrast, STAT phosphorylation was readily detectedin cells exposed to HS3, which lacks the aMTD motif required formembrane penetration, indicating that HS3, which lacks an MTD sequencedid not enter the cells, has no biological activity.

Example 8-2. Biological Activity Test of iCP-SOCS3

Peritoneal macrophages were obtained from C3H/HeJ mice (Doo-Yeol BiotechCo. Ltd. Korea) Peritoneal macrophages were incubated with 10 μM SOCS3recombinant proteins (1:HS3, 2:HM₁₆₅S3, 3:HM₁₆₅S3A and 4:HM₁₆₅S3B,respectively) for 1 hr at 37° C. and then stimulated them with LPS(Lipopolysaccharide)(500 ng/ml) and/or IFN-γ (100 U/ml) without removingiCP-SOCS3 proteins for 3, 6, or 9 hrs. The culture media were collected,and the extracellular levels of cytokine (TNF-α, IL-6) were measured bya cytometric bead array (BD Pharmingen, San Diego, Calif., USA)according to the manufacturer's instructions.

The effect of cell-permeable SOCS3 proteins on cytokines secretion wasinvestigated. Treatment of C3H/HeJ primary peritoneal macrophages withSOCS3 proteins containing aMTD₁₆₅ suppressed TNF-α and IL-6 secretioninduced by the combination of IFN-γ and LPS by 50-90% during subsequent9 hrs of incubation (FIG. 24). In particular, aMTD₁₆₅/SDB-fused SOCS3recombinant protein showed the greatest inhibitory effect on cytokinesecretion. In contrast, cytokine secretion in macrophages treated withnon-permeable SOCS3 protein (HS3) was unchanged, indicating thatrecombinant SOCS3 lacking the aMTD doesn't affect intracellularsignaling. Therefore, we conclude that differences in the biologicalactivities of HM₁₆₅S3B as compared to HS3B are due to the differences inprotein uptake mediated by the aMTD sequence. In light ofsolubility/yield, cell-/tissue-permeability, and biological effect,SOCS3 recombinant protein containing aMTD and SDB (HM₁₆₅S3B) is aprototype of a new generation of improved cell-permeable SOCS3(iCP-SOCS3), and will be selected for further evaluation as a potentialanti-tumor agent.

Example 9. Preparation of Control Protein (Non-CP-SOCS3: HS3BRecombinant Protein)

As an experimental negative control, a SOCS3 recombinant protein havingno cell permeability was prepared.

According to example 6-2, SOCS3 recombinant proteins lacking SD(HM₁₆₅S3) or both aMTD and SD (HS3) were found to be less soluble,produced lower yields, and showed tendency to precipitate when they wereexpressed and purified in E. coli. Therefore, we additionally designedand constructed SOCS3 recombinant protein containing only SDB (withoutaMTD₁₆₅: HS3B) as a negative control (FIG. 25). Preparation, expressionand purification, and measurement of solubility/yield of the recombinantproteins were performed in the same manner as in Examples 6-2 and 6-3.

TABLE 37 Recombinant Cargo SD Protein 5′-primers 3′-pimers SOCS3 B HS3B5′-GGAATTCCATATGGTCACCCACA 5′-CGCGTCGACTTAAAGGGT GCAAGTTTCCCGCCGCC-3′TTCCGAAGGCTTGGCTATCT (SEQ ID NO: 969) T-3′ (SEQ ID NO: 970)

As expected, its solubility and yield increased compared to that ofSOCS3 proteins lacking SDB (HS3; FIG. 26). Therefore, HS3B proteins wereused as a control protein.

Example 10. Selection of aMTD for Cell-Permeability

After a basic structure of the stable recombinant proteins fused withcombinations of aMTD and SD was determined, 22 aMTDs were selected fordevelopment of iCP-SOCS3 recombinant protein (FIGS. 82 and 83), based onthe critical Factors, in order to examine which aMTD provides thehighest cell-/tissue-permeability.

For comparison, 5 kinds of random peptides that do not satisfying one ormore critical factors were selected (FIG. 84).

Solubility/yield and cell-permeability of 22 kinds of aMTD/SDB-fusedSOCS3 recombinant proteins, prepared by using primers of Table 38 in thesame manner as in Example 6-2, were analyzed according to examples 6-3and 7-1, respectively.

Amino Acid aMTD Sequence Cargo ID (SEQ ID NO) 5′ Primers 3′ PrimersSOCS3 MTM AAVLLPVLLAAP GGAATTCCATATGGCGGCGGTGCTGCTGCCG CGCGTCGACTT(SEQ ID NO: 865) GTGCTGCTGGCGGCGCCGGTCACCCACAGC AAAGGGTTTCCAAGTTTCCCGCCGCC GAAGGCTTGGC (SEQ ID NO: 977) TATCTT 44 ALAVPVALLVAPGGAATTCCATATGGCGCTGGCGGTGCCGGTG (SEQ ID NO:  (SEQ ID NO: 16)GCGCTGCTGGTGGCGCCGGTCACCCACAGCA 864) AGTTTCCCGCCGCC (SEQ ID NO: 978) 81AALLPALAALLP GGAATTCCATATGGCGGCGCTGCTGCCGGCG (SEQ ID NO: 22)CTGGCGGCGCTGCTGCCGGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 979) 123AAIIVPAALLAP GGAATTCCATATGGCGGCGATTATTGTGCC (SEQ ID NO: 33)GGCGGCGCTGCTGGCGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 980) 162AVVALPAALIVP GGAATTCCATATGGCGGTGGTGGCGCTGCCG (SEQ ID NO: 40)GCGGCGCTGATTGTGCCGGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 981) 281ALIVLPAAVAVP GGAATTCCATATGGCGCTGATTGTGCTGCC (SEQ ID NO: 65)GGCGGCGGTGGCGGTGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 982) 324IVAVALPAALVP GGAATTCCATATGATTGTGGCGGTGGCGCT (SEQ ID NO: 77)GCCGGCGGCGCTGGTGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 983) 364LVAAVAPALIVP GGAATTCCATATGCTGGTGGCGGCGGTGGCG (SEQ ID NO: 85)CCGGCGCTGATTGTGCCGGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 839) 365AVIVVAPALLAP GGAATTCCATATGGCGGTGATTGTGGTGGC (SEQ ID NO: 86)GCCGGCGCTGCTGGCGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 840) 622ALIVLAAPVAVP GGAATTCCATATGGCGCTGATTGTGCTGGC (SEQ ID NO: 142)GGCGCCGGTGGCGGTGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 841) 662ALAVILAPVAVP GGAATTCCATATGGCGCTGGCGGTGATTCT (SEQ ID NO: 822)GGCGCCGGTGGCGGTGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 842) 563ALAVIVVPALAP GGAATTCCATATGGCGCTGGCGGTGATTGT (SEQ ID NO: 131)GGTGCCGGCGCTGGCGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 843) 899AVVIALPAVVAP GGAATTCCATATGGCGGTGGTGATTGCGCT (SEQ ID NO: 229)GCCGGCGGTGGTGGCGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 844) 897AVIVPVAIIAAP GGAATTCCATATGGCGGTGATTGTGCCGGT (SEQ ID NO: 228)GGCGATTATTGCGGCGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 845) 623VAAAIALPAIVP GGAATTCCATATGGTGGCGGCGGCGATTGCG (SEQ ID NO: 143)CTGCCGGCGATTGTGCCGGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 846) 908VALALAPVVVAP GGAATTCCATATGGTGGCGCTGGCGCTGGCG (SEQ ID NO: 237)CCGGTGGTGGTGGCGCCGGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 848) 911VALALPAVVVAP GGAATTCCATATGGTGGCGCTGGCGCTGCCG (SEQ ID NO: 239)GCGGTGGTGGTGGCGCCGGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 849) 2AAAVPLLAVVVP GGAATTCCATATGGCGGCGGCGGTGCCGCTG (SEQ ID NO: 2)CTGGCGGTGGTGGTGCCGGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 850) 904AVLAVVAPVVAP GGAATTCCATATGGCGGTGCTGGCGGTGGTG (SEQ ID NO: 233)GCGCCGGTGGTGGCGCCGGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 851) 481AIAIAIVPVALP GGAATTCCATATGGCGATTGCGATTGCGAT (SEQ ID NO: 110)TGTGCCGGTGGCGCTGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 852) 787AVALVPVIVAAP GGAATTCCATATGGCGGTGGCGCTGGTGCCG (SEQ ID NO: 177)GTGATTGTGGCGGCGCCGGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 853) 264LAAAPVVIVIAP GGAATTCCATATGCTGGCGGCGGCGCCGGTG (SEQ ID NO: 63)GTGATTGTGATTGCGCCGGTCACCCACAGC AAGTTTCCCGCCGCC (SEQ ID NO: 854) 363AVLAVAPALIVP GGAATTCCATATGGCGGTGCTGGCGGTGGCG (SEQ ID NO: 84)CCGGCGCTGATTGTGCCGGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 855) 121AIVALPALALAP GGAATTCCATATGGCGATTGTGGCGCTGCC (SEQ ID NO: 32)GGCGCTGGCGCTGGCGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 856) 921IWWFVVLPLVVP GGAATTCCATATGATTTGGTGGTTTGTGGT (SEQ ID NO: 931)GCTGCCGCTGGTGGTGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 857) 16NNSCTTYTNGSQ GGAATTCCATATGAACAACAGCTGCACCACC (SEQ ID NO: 890)TATACCAACGGCAGCCAGGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 858) 67LDAEVPLADDVP GGAATTCCATATGCTGGATGCGGAAGTGCCG (SEQ ID NO: 906)CTGGCGGATGATGTGCCGGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 861) 29VLPPLPVLPVLP GGAATTCCATATGGTGCTGCCGCCGCTGCC (SEQ ID NO: 924)GGTGCTGCCGGTGCTGCCGGTCACCCACAG CAAGTTTCCCGCCGCC (SEQ ID NO: 862) 700GTSNTCQSNQNS GGAATTCCATATGGGCACCAGCAACACCTGC (SEQ ID NO: 951)CAGAGCAACCAGAACAGCGTCACCCACAGCA AGTTTCCCGCCGCC (SEQ ID NO: 863)

As shown in FIGS. 27 to 34, it was confirmed that most of theaMTD/SDB-fused SOCS3 recombinant proteins showed high solubility andyield and high cell permeability by aMTD. However, Randompeptide-SOCS3-SDB recombinant protein showed remarkably low cellpermeability.

Example 11-1. Investigation of Biological Activity for Determination ofOptimal aMTD-Fused SOCS3 Recombinant Protein-1

Four kinds of aMTD/SD-fused SOCS3 recombinant proteins having high cellpermeability and one kind of aMTD/SD-fused SOCS3 recombinant proteinhaving the lowest cell permeability were selected, and their biologicalactivity was analyzed.

PANC-1 cells (pancreatic carcinoma cell line) were seeded in a 16-wellchamber slide at a density of 5×10³ cells/well, and then treated with 10uM of aMTD/SD-fused SOCS3 for 24 hours. Apoptotic cells were analyzedusing terminal dUTP nick-end labeling (TUNEL) assay with In Situ CellDeath Detection kit TMR red (Roche, 4056 Basel, Switzerland). Cells weretreated with either 10 μM SOCS3 recombinant protein or buffer alone for16 hrs with 2% fetal bovine serum (Hyclone, Logan, Utah, USA). Treatedcells were washed with cold PBS two times, fixed in 4% paraformaldehyde(PFA, Junsei, Tokyo, Japan) for 1 hr at room temperature, and incubatedin 0.1% Triton X-100 for 2 min on the ice. Cells were washed with coldPBS twice, and treated TUNEL reaction mixture for 1 hr at 37° C. indark, washed cold PBS three times and observed by fluorescencemicroscopy (Nikon, Tokyo, Japan).

As shown in FIG. 35, most of the aMTD/SDB-fused SOCS3 recombinantproteins induced cell death of pancreatic carcinoma cells, and of them,aMTD₁₆₅ or aMTD324-fused SOCS3 recombinant protein induced death of thelargest number of cancer cells.

Example 11-2. Investigation of Biological Activity for Determination ofOptimal aMTD-Fused SOCS3 Recombinant Protein-2

AGS cells (gastric carcinoma cell line) (American Type CultureCollection; ATCC) were seeded in a 12-well plate at a density of 1×10⁵cells/well, and then treated with 10 uM of aMTD/SD-fused SOCS3 for 14hours. Cancer cell death was analyzed by Annexin V analysis. AnnexinV/7-Aminoactinomycin D (7-AAD) staining was performed using flowcytometry according to the manufacturer's guidelines (BD Pharmingen, SanDiego, Calif., USA). Briefly, cells were washed three times withice-cold PBS. The cells were then resuspended in 100 μl of bindingbuffer and incubated with 1 μl of 7-AAD and 1 μl of annexin V-PE for 30min in the dark at 37° C. Flow cytometric analysis was immediatelyperformed using a guava easyCyte™ 8 Instrument (Merck Millipore,Darmstadt, Germany).

As shown in FIG. 36, most of the aMTD/SDB-fused SOCS3 recombinantproteins induced cell death of gastric carcinoma cells, and of them,aMTD₁₆₅ or aMTD₂₈₁-fused SOCS3 recombinant protein induced death of thelargest number of cancer cells.

Example 11-3. Investigation of Biological Activity for Determination ofOptimal aMTD-Fused SOCS3 Recombinant Protein-3

AGS cells (gastric cancer cell line) were seeded in a 12 well plate at adensity of 2.5×10⁵ per well, grown to 90% confluence. Confluent AGScells were incubated with 10 μM HM # S3B in serum-free medium for 2 hrsprior to changing the growth medium (DMEM/F12, Hyclone, Logan, Utah,USA) and washed twice with PBS, and the monolayer at the center of thewell was “wounded” by scraping with a sterilized white pipette tip.Cells were cultured for an additional 24 hrs and cell migration wasobserved by phase contrast microscopy (Nikon, ECLIPSE Ts2). Themigration was quantified by counting the number of cells that migratedfrom the wound edge into the clear area.

As shown in FIG. 37, most of the aMTD/SDB-fused SOCS3 recombinantproteins inhibited cell migration of gastric carcinoma cells, and ofthem, aMTD₁₆₅ or aMTD₉₀₄-fused SOCS3 recombinant protein showed the mosteffective inhibition of cancer cell migration.

Solubility/yield, permeability, and biological activity of 22 kinds ofthe aMTD-fused recombinant proteins were examined in Examples 10 to11-3, and as a result, the aMTD₁₆₅/SDB-fused SOCS3 recombinant proteinwas found to show the most excellent effect (FIG. 38). Therefore, theaMTD₁₆₅-fused recombinant protein was used in the subsequent experiment.

Example 12-1. Preparation of iCP-SOCS3 Recombinant Protein andInvestigation of Equivalence Thereof-1

To develop a new drug as an anticancer agent, His-tag-removed iCP-SOCS3recombinant protein was prepared and equivalence of His-Tag+, −iCP-SOCS3was investigated.

Histidine-tag free human SOCS3 proteins were constructed by amplifyingthe SOCS3 cDNA (225 amino acids) for aMTD fused to SOCS3 cargo. The PCRreactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTPmixture, 1× reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctorprotein, Korea)) were digested on the restriction enzyme site betweenNde I (5′) and Sal I (3′) involving 35 cycles of denaturing (95° C.),annealing (62° C.), and extending (72° C.) for 45 sec each. For the lastextension cycle, the PCR reactions remained for 10 min at 72° C. The PCRproducts were subcloned into pET-26b(+) (Novagen, Darmstadt, Germany).Coding sequence for SDB fused to C terminus of aMTD-SOCS3 was cloned atBamHI (5′) and SalI (3′) in pET-26b(+) from PCR-amplified DNA segmentsand confirmed by DNA sequence analysis of the resulting plasmids.

Recombinant Cargo SD Protein 5′ Primers 3′ Primers SOCS3 — HS35′-GGAATTCCATATGGTCAC 5′-CCCGGATCCTTAAAGC CCACAGCAAGTTTCCCGCCGGGGCATCGTACTGGTCC GCC-3′ (SEQ ID NO: 955) AGGAA-3′(SEQ ID NO: 956) —HM₁₆₅S3 5′-GGAATTCCATATGGCGCT 5′-CCCGGATCCTTAAAGC GGCGGTGCCGGTGGCGCTGGGGGCATCGTACTGGTCC GCGATTGTGCCGGTCACCC AGGAA-3′ (SEQ ID NO: 958)ACAGCAAGTTTC-3′ (SEQ ID NO: 957) A HM₁₆₅S3A 5′-GGAATTCCATATGGCGCT5′-CGCGTCGACTTACCTCGG GGCGGTGCCGGTGGCGCTG CTGCACCGGCACGGCGATGCGATTGTGCCGGTCACCC AC-3′ (SEQ ID NO: 960) ACAGCAAGTTTC-3′(SEQ ID NO: 959) B EM₁₆₅S3B 5′-GGAATTCCATATGGCGCT 5′-CGCGTCGACTTAAAGGGGCGGTGCCGGTGGCGCTG GTTTCCGAAGGCTTGGCT GCGATTGTGCCGGTCACCCATCTT-3′ (SEQ ID NO: 962) ACAGCAAGTTTC-3′ (SEQ ID NO: 961) C EM₁₆₅S3C5′-GGAATTCCATATGGCGCT 5′-GCGTCGACTTAGGCCAGG GGCGGTGCCGGTGGCGCTGTTAGCGTCGAG-3′ GCGATTGTGCCGGTCACCC (SEQ ID NO: 964) ACAGCAAGTTTC-3′(SEQ ID NO: 963) D EM₁₆₅S3D 5′-GGAATTCCATATGGCGCT 5′-GCGTCGACTTATTTTTTCTGGCGGTGCCGGTGGCGCTG CGGACAGATA-3′ GCGATTGTGCCGGTCACCC (SEQ ID NO: 966)ACAGCAAGTTTC-3′ (SEQ ID NO: 965) E EM₁₆₅S3E 5′-GGAATTCCATATGGCGCT5′-ACGCGTCGACTTAACCT GGCGGTGCCGGTGGCGCTG CCAATCTGTTCGCGGTGAGCGATTGTGCCGGTCACCC GCCTC-3′ (SEQ ID NO: 968) ACAGCAAGTTTC-3′(SEQ ID NO: 967) B M₁₆₅S3B 5′-GGAATTCCATATGGCGCT 5′-CGCGTCGACTTAAAGGGGCGGTGCCGGTGGCGCTG GTTTCCGAAGGCTTGGCT GCGATTGTGCCGGTCACCC ATCTT-3′ACAGCAAGTTTC-3′ (SEQ ID NO: 860) (SEQ ID NO: 859)

Expression, purification and solubility/yield were measured in the samemanner as in Examples 6-2 and 6-3, and as a result, his-tag-removedM₁₆₅S3B was found to have high solubility/yield (FIG. 39b ).

Example 12-2. Preparation of iCP-SOCS3 Recombinant Protein andInvestigation of Equivalence Thereof-2

In the same manner as in Example 7-1, RAW264.7 cells were treated withFITC-labeled HS3B, HM₁₆₅S3B, and M₁₆₅ S3B proteins, and cellpermeability was evaluated.

As shown in FIG. 40, both HM₁₆₅S3B and M₁₆₅ S3B were found to have highcell permeability.

Example 12-3. Preparation of iCP-SOCS3 Recombinant Protein andInvestigation of Equivalence Thereof-3

To investigate biological activity equivalence of the HM₁₆₅S3B andM₁₆₅S3B recombinant proteins, induction of apoptosis of gastriccarcinoma cell line (AGS) was analyzed by Annexin V staining in the samemanner as in Example 11-2, and inhibition of migration was analyzed inthe same manner as in Example 11-3.

As shown in FIG. 41, it was confirmed that both HM₁₆₅S3B and M₁₆₅S3Bshowed high anticancer efficacy and M₁₆₅S3B exhibited efficacyequivalent to or higher than HM₁₆₅S3B.

Example 12-4. Preparation of iCP-SOCS3 Recombinant Protein andInvestigation of Equivalence Thereof-4

In silico MHC class II binding analysis using iTope™ (ANTITOPE.LTD)revealed changing the V28 pl anchor residue in SDB sequence to L makesthis region human germline and as such both MHC class II bindingpeptides within this region would be expected to be low risk due to Tcell tolerance.

To prepare humanized SDB domain, iCP-SOCS3 was prepared as in FIG. 43.iCP-SOCS3 was prepared in the same manner as in Example 6-1, except thatSDB′ having a substitution of valine with leucine at an amino acidposition 28 was used (FIG. 70). Further, protein purification wasperformed in the same manner as in example 6-2 using the primer as below(Tables 39 and 40).

TABLE 40 Recombinant Cargo SD Protein 5′ Primers 3′ Primers SOCS3 B*HM₁₆₅S3B* 5′-GGAATTCCATATGGC 5′-CGCGTCGACTTAAAGGG GCTGGCGGTGCCGGTGGTTTCCGAAGGCTTGGCTATC CGCTGGCGATTGTGCCGG TT-3′ TCACCCACAGCAAGTTTC-3′(SEQ ID NO: 972) (SEQ ID NO: 971)

As shown in FIG. 44, both HM₁₆₅S3B and HM₁₆₅S3B′(V28L) were found tohave high solubility/yield.

Example 12-5. Preparation of iCP-SOCS3 Recombinant Protein andInvestigation of Equivalence Thereof-5

In the same manner as in Example 7-1, RAW264.7 cells were treated withFITC-labeled HM₁₆₅S3B and HM₁₆₅S3B′(V28L) proteins, and cellpermeability was evaluated.

As shown in FIG. 45, both HM₁₆₅S3B and HM₁₆₅S3B′(V28L) were found tohave high cell permeability.

Example 12-6. Preparation of iCP-SOCS3 Recombinant Protein andInvestigation of Equivalence Thereof-6

To investigate biological activity equivalence of the HM₁₆₅S3B andHM₁₆₅S3B′(V28L) recombinant proteins, anti-proliferative activity wasexamined and induction of apoptosis of gastric carcinoma cell line (AGS)was analyzed by Annexin V staining in the same manner as in Example11-2, and inhibition of migration was analyzed in the same manner as inExample 11-3.

Antiproliferative activity were evaluated with the CellTiter-Glo CellViability Assay. AGS cells (3×10³/well) were seeded in 96 well plates.The next day, cells were treated with DMEM (vehicle) or 10 μM HM₁₆₅S3B,HM₁₆₅S3B′(V28L) for 96 hrs in the presence of serum (2%). Proteins werereplaced daily. Cell growth and survival were evaluated with theCellTiter-Glo Cell Viability Assay (Promega, Madison, Wis.).Measurements using a Luminometer (Turner Designs, Sunnyvale, Calif.)were conducted following the manufacturer's protocol.

It was confirmed that both HM₁₆₅S3B and HM₁₆₅S3B′(V28L) showed highanti-proliferative effects on gastric carcinoma cells (FIG. 46), andalso effects of inducing apoptosis (FIG. 47) and of inhibiting migrationof gastric carcinoma cells (FIGS. 48a and 48b ), and in particular,HM₁₆₅S3B′(V28L) exhibited efficacy equivalent to or higher thanHM₁₆₅S3B.

Example 12-7. Preparation of iCP-SOCS3 Recombinant Protein andInvestigation of Equivalence Thereof-7

iCP-SOCS3 of BS3M₁₆₅ structure was prepared in the same manner as inExample 6-1, and B′S3M₁₆₅ iCP-SOCS3 was also prepared by humanized SDBdomain (FIG. 49a ).

TABLE 41 Recombinant Cargo SD Protein 5′ Primers 3′ Primers SOCS3 BBS3M₁₆₅ 5′-GGAATTCCATATGAT 5′-ACGCGTCGACTTACGCCAG GGCAGAACAAAGCGAC-3′CGCCACCGGCACCGCCAGCGC (SEQ ID NO: 973) AATCACCGGAAGCGGGGCATCGTACTGGTCCAG-3′ (SEQ ID NO: 974) B* B*S3M₁₆₅ 5′-GGAATTCCATATGAT5′-ACGCGTCGACTTACGCCAG GGCAGAACAAAGCGAC- CGCCACCGGCACCGCCAGCGC 3′AATCACCGGAAGCGGGGCATC (SEQ ID NO: 975) GTACTGGTCCAG-3′ (SEQ ID NO: 976)

Expressions and purifications of iCP-SOCS3 recombinant protein (BS3M₁₆₅,B′S3M₁₆₅) in E. coli were analyzed in the same manner as in Examples 6-2and 6-3, respectively, and were shown in FIGS. 49b and 49c . Further, E.coli codon-optimized iCP-SOCS3 was prepared.

Example 13. Test of Biological Activity of iCP-SOCS3-Inhibition Activityof IFN-γ-Induced STAT Phosphorylation

Whether iCP-SOCS3 (HM₁₆₅S3B) recombinant protein inhibits activation ofthe JAK/STAT-signaling pathway was examined by the method of Example8-1.

PANC-1 Cells (KCLB, Seoul, South Korea) were treated with 10 ng/ml IFN-γ(R&D systems, Abingdon, UK) for 15 min and treated with either DMEM(vehicle) or 1, 5, 10 μM aMTD/SD-fused SOCS3 recombinant proteins for 2hrs. The cells were lysed in RIPA lysis buffer (Biosesang, Seongnam,Korea) containing proteinase inhibitor cocktail (Roche, Indianapolis,Ind., USA), incubated for 15 min at 4° C., and centrifuged at 13,000 rpmfor 10 min at 4° C. Equal amounts of lysates were separated on 10%SDS-PAGE gels and transferred to a nitrocellulose membrane. Themembranes were blocked using 5% skim milk in TBST and for western blotanalysis incubated with the following antibodies: anti-phospho-STAT3(Cell Signaling Technology, USA), then HRP conjugated anti-rabbitsecondary antibody (Santacruz).

The well-known cytokine signaling inhibitory actions of SOCS3 areinflammation inhibition through i) inhibition of IFN-γ-mediated JAK/STATand ii) inhibition of LPS-mediated cytokine secretion. The ultimate testof cell-penetrating efficiency is a determination of intracellularactivity of SOCS3 proteins transported by aMTD. Since endogenous SOCS3are known to block phosphorylation of STAT3 by IFN-γ-mediated Januskinases (JAK) 1 and 2 activation, we demonstrated whether cell-permeableSOCS3 inhibits the phosphorylation of STATs. As shown in FIG. 50,iCP-SOCS3 (HM₁₆₅S3B) suppressed IFN-γ-induced phosphorylation of STAT3in dose dependent manner. In contrast, STAT phosphorylation was readilydetected in cells exposed to HS3B, which lacks the aMTD motif requiredfor membrane penetration, indicating that HS3B, which lacks an MTDsequence did not enter the cells, has no biological activity.

Example 14-1. Investigation of aMTD-Mediated Intracellular DeliveryMechanism

The mechanism of aMTD₁₆₅-mediated intracellular delivery wasinvestigated.

(1) RAW 264.7 cells were pretreated with 100 mM EDTA for 3 hours, andthen treated with 10 uM of iCP-SOCS3 (HM₁₆₅S3B) protein for 1 hour,followed by flow cytometry in the same manner as in Example 7-1 (FIG.51a ).

(2) RAW 264.7 cells were pretreated with 5 ug/ml of proteinase K for 10minutes, and then treated with 10 uM of iCP-SOCS3 (HM₁₆₅S3B) protein for1 hour, followed by flow cytometry (FIG. 51b ).

(3) RAW 264.7 cells were pretreated with 20 uM taxol for 30 minutes, andthen treated with 10 uM of iCP-SOCS3 (HM₁₆₅S3B) protein for 1 hour,followed by flow cytometry (FIG. 52a ).

(4) RAW 264.7 cells were pretreated with 1 mM ATP and 10 uM antimycinsingly or in combination for 2 hours, and then treated with 10 uM ofiCP-SOCS3 (HM₁₆₅S3B) protein for 1 hour, followed by flow cytometry(FIG. 52b ).

(5) RAW 264.7 cells were left at 4° C. and 37° C. for 1 hour,respectively, and then treated with 10 uM of iCP-SOCS3 (HM₁₆₅S3B)protein for 1 hour, followed by flow cytometry (FIG. 53).

The aMTD-mediated intracellular delivery of SOCS3 protein did notrequire protease-sensitive protein domains displayed on the cell surface(FIG. 51b ), microtubule function (FIG. 52a ), or ATP utilization (FIG.52b ), since aMTD₁₆₅-dependent uptake, compare to HS3 and HS3B, wasessentially unaffected by treating cells with proteinase K, taxol, orthe ATP depleting agent, antimycin. Conversely, iCP-SOCS3 (HM₁₆₅S3B)proteins uptake was blocked by treatment with EDTA and low temperature(FIGS. 51a and 53), indicating the importance of membrane integrity andfluidity for aMTD-mediated protein transduction.

Moreover, whether cells treated with iCP-SOCS3 (HM₁₆₅S3B) protein couldtransfer the protein to neighboring cells were also tested.

For this, RAW 264.7 cells were treated with 10 uM of FITC-labelediCP-SOCS3 (HM₁₆₅S3B) protein for 1 hour. Thereafter, these cells wereco-cultured with PerCP-Cy5.5-CD14-stained RAW 264.7 cells for 2 hours.Cell-to-cell protein transfer was assessed by flow cytometry, scoringfor CD14/FITC double-positive cells. Efficient cell-to-cell transfer ofHM₁₆₅S3B, but not HS3 or HS3B (FIG. 54), suggests that SOCS3 recombinantproteins containing aMTD165 are capable of bidirectional passage acrossthe plasma membrane.

Example 14-2. Investigation of Cationic CPP-Mediated IntracellularDelivery Mechanism

The mechanism of basic CPP (TAT and PolyR)-mediated intracellulardelivery was also investigated in the same manner as in Example 7-1 andExample 14-1.

As shown in FIGS. 75a and 75b , it was confirmed that aMTD165/SD-fusedSOCS3 recombinant proteins are independent to cell surface receptor(FIG. 75a ) and the cell-permeability of aMTD165/SD-fused SOCS3recombinant proteins is not due to endocytosis (FIG. 75b ).

Whether cells treated with aMTD165/SD-fused SOCS3, TAT/SD-fused SOCS3,and PolyR/SD-fused SOCS3 could transfer the protein to neighboring cellswere also tested on a molecular level in the same manner as in Example13.

For this, RAW 264.7 cells were treated with 5 μM of FITC-labeledHM₁₆₅S3B, HTS3B for 2 hour and washed with PBS two times. Thereafter,they were seeded on PANC-1 cell, incubated for 2 hours and treated with20 ng/ml of IFN-γ for 15 minutes, followed by Western blotting in thesame manner as in Example 8-1. And Cell-to-cell protein transfer wasassessed by flow cytometry.

As shown in FIG. 76, efficient cell-to-cell transfer of HM₁₆₅S3B, butnot HTS3B or HRS3B, suggests that only SOCS3 recombinant proteinscontaining aMTD165 are capable of bidirectional passage across theplasma membrane.

Moreover, as shown in FIG. 77, phospho-STAT3 was only reduced in cellstreated with HM₁₆₅S3B.

Example 14-3. Investigation of Time- and Dose-Dependency of iCP-SOCS3Cell-Permeability

It was examined whether the cell-permeability of iCP-SOCS3 recombinantprotein is dose-dependent. Cell-permeability of iCP-SOCS3 recombinantprotein was tested in the same manner as in Example 7-1 except for thecells being treated with 0.05 μM-10 μM of iCP-SOCS3 recombinant proteinfor 1 hrs. As shown in FIGS. 78a and 78b , it was confirmed that thecell-permeability of iCP-SOCS3 recombinant protein is dose-dependent

Time-dependency of cell-permeability was also investigated. The cellswere treated with 10 μM of iCP-SOCS3 recombinant protein for 5-180minutes. As shown in FIGS. 79a and 79b , high level of cell-permeabilityof iCP-SOCS3 was observed only 5 minutes post-treatment and could beseen even at 180 minutes mark.

Example 15. Investigation of Bioavailability of iCP-SOCS3

To investigate BA of the iCP-SOCS3 (HM₁₆₅S3B) recombinant proteins, ICRmice (Doo-Yeol Biotech Co. Ltd., Seoul, Korea) were intravenous (IV)injected with 600 ug/head of 10 μM FITC (Fluorescein isothiocyanate,SIGMA, USA)-labeled SOCS3 recombinant proteins (HS3B, HM₁₆₅S3B) andafter 15 min, 30 min, 1H, 2H, 4H, 8H, 12H, 16H, 24H, 36H, 48H, mice ofeach group were sacrificed. From the mice, peripheral blood mononuclearcells (PBMCs), splenocytes, and hepatocytes were separated.

Further, the spleen was removed and washed with PBS, and then placed ona dry ice and embedded in an O.C.T. compound (Sakura). Aftercryosectioning at 20 μm, tissue distributions offluorescence-labeled-SOCS3 proteins in different organs was analyzed byfluorescence microscopy (Carl Zeiss, Gottingen, Germany).

Isolation of PBMC

After anesthesia with ether, ophthalmectomy was performed and the bloodwas collected therefrom using a 1 ml syringe. The collected blood wasimmediately put in an EDTA tube and mixed well. The blood wascentrifuged at 4,000 rpm and 4° C. for 5 minutes, and plasma wasdiscarded and only buffy coat was collected in a new microtube. 0.5 mlof RBC lysis buffer (Sigma) was added thereto, followed by vortexing.The microtube was left at room temperature for 5 minutes, and thencentrifuged at 4,000 rpm and 4° C. for 5 minutes. 0.3 ml of PBS wasadded to a pellet, followed by pipetting and flow cytometry (FlowJocytometric analysis software, Guava, Millipore, Darmstadt, Germany).

Isolation of Splenocytes and Hepatocytes

Mice were laparotomized and the spleen or liver were removed. The spleenor liver thus removed was separated into single cells using a CellStrainer (SPL, Korea). These cells were collected in a microtube,followed by centrifugation at 4,000 rpm and 4° C. for 5 minutes. 0.5 mlof RBC lysis buffer was added thereto, followed by vortexing. Themicrotube was left at room temperature for 5 minutes, and thencentrifuged at 4,000 rpm and 4° C. for 5 minutes. 0.5 ml of PBS wasadded to a pellet, followed by pipetting and flow cytometry (FlowJocytometric analysis software, Guava, Millipore, Darmstadt, Germany).

As shown in FIG. 55, in PBMCs, the maximum permeability of iCP-SOCS3 wasobserved at 30 minutes, and in splenocytes, the maximum permeability ofiCP-SOCS3 was observed at 2 hours and maintained up to 16 hours. Inhepatocytes, the maximum permeability of iCP-SOCS3 was observed at 15minutes and maintained up to 16 hours.

Example 16. Investigation of Bio-Distribution of iCP-SOCS3 RecombinantProtein

To investigate BA of the iCP-SOCS3 (HM₁₆₅S3B) recombinant proteins, ICRmice (Doo-Yeol Biotech Co. Ltd., Seoul, Korea) were intravenous (IV)injected with 600 rig/head of 10 μM FITC (Fluorescein isothiocyanate,SIGMA, USA)-labeled SOCS3 proteins (HS3B, HM₁₆₅S3B) and after 2H, 8H,12H, 24H, mice of each group were sacrificed. From the mice, pancreaswas removed and washed with PBS, and then placed on a dry ice andembedded in an O.C.T. compound (Sakura). After cryosectioning at 20 μm,tissue distributions of fluorescence-labeled-SOCS3 proteins in pancreastissue was analyzed by fluorescence microscopy (Carl Zeiss, Gottingen,Germany) (FIG. 56).

As shown in FIG. 56, in the pancreas tissue, very high distribution ofiCP-SOCS3 was observed at 2 hours, and maintained up to 8 hours.Therefore, it can be seen that iCP-SOCS3 is rapidly delivered from bloodto various tissues within 2 hours, and maintained up to 8-16 hoursdepending on the tissues.

Example 17-1. Investigation of Angiogenesis-Inhibitory Efficacy ofiCP-SOCS3 Recombinant Protein: Cell Proliferation

Inhibition of growth of vascular endothelial cells by iCP-SOCS3 wasanalyzed. HUVECs were purchased (ATCC, Manassas, Va.) and maintained asrecommended by the supplier. These cells (3×10³/well) were seeded in 96well plates. The next day, cells were treated with DMEM (vehicle) orrecombinant SOCS3 proteins for 96 hrs in the presence of serum (2%).Proteins were replaced daily. Cell growth and survival were evaluatedwith the CellTiter-Glo Cell Viability Assay (Promega, Madison, Wis.).Measurements using a Luminometer (Turner Designs, Sunnyvale, Calif.)were conducted following the manufacturer's protocol (FIG. 57). As shownin FIG. 57, it was confirmed that HM₁₆₅S3B significantly inhibitedviability of vascular endothelial cells.

Example 17-2. Investigation of Angiogenesis-Inhibitory Efficacy ofiCP-SOCS3 Recombinant Protein: Cell Migration

Inhibition of migration of vascular endothelial cells by iCP-SOCS3 wasanalyzed. The lower surface of Transwell inserts (Costar) was coatedwith 0.1% gelatin, and the membranes were allowed to dry for 1 hr atroom temperature. The Transwell inserts were assembled into a 24-wellplate, and the lower chamber was filled with growth media containing 10%FBS and FGF2 (40 ng/ml). Cells (5×10⁵) were added to each upper chamber,and the plate was incubated at 37° C. in a 5% CO₂ incubator for 24 hrs.Migrated cells were stained with 0.6% hematoxylin and 0.5% eosin andcounted. (FIG. 59). HM₁₆₅S3B significantly inhibited migration ofvascular endothelial cells.

Example 17-3. Investigation of Angiogenesis-Inhibitory Efficacy ofiCP-SOCS3 Recombinant Protein: Tube Formation

Inhibition of angiogenesis of vascular endothelial cells by iCP-SOCS3was analyzed. Endothelial cell tube-forming assays are performed in24-well Matrigel (BD Bioscience) coated plates. Human umbilical-veinendothelial cells (HUVECs) are treated with 10 μM of recombinant SOCS3protein for 8 hrs and then the trypsinized cells were seeded on thesurface of the Matrigel (5×10⁴/well). Tube formation is monitored after24 hrs and the images were analyzed using a service provided by Wimasis.(FIG. 59). HM₁₆₅S3B significantly inhibited the angiogenic ability ofvascular endothelial cells.

Example 17-4. Investigation of Angiogenesis-Inhibitory Efficacy ofiCP-SOCS3 Recombinant Protein: Molecular Mechanism

The mechanism of angiogenesis inhibition by iCP-SOCS3 recombinantprotein was investigated. HUVEC cells were seeded at a density of 5×10⁵in a 6-well plate, and on next day, the cells were starved for 2 hours,followed by treatment of SOCS3 recombinant proteins for 2 hours. Lastly,the cells were treated with bFGF (40 ng/mL) for 2 hours, and then cellswere harvested. Proteins were isolated from the cells using an RIPAlysis buffer (Biosesang, Seongnam, Korea), and 20 μg thereof was used toperform Western blot analysis. As shown in FIG. 60, it was investigatedthat angiogenesis mechanism (mTOR/Akt pathway) was inhibited by theiCP-SOCS3 recombinant proteins.

Example 17-5. Investigation of Angiogenesis-Inhibitory Efficacy ofiCP-SOCS3 Recombinant Protein: Chick Embryonic Aortic Ring Assay

In order to investigate a mechanism-specific angiogenesis-inhibitoryefficacy of the iCP-SOCS3 recombinant protein, an Ex vivo experimentalmodel was employed, and inhibition of angiogenesis required forembryonic development was investigated by a chick embryonic aortic ringassay.

Chicken fertilized eggs were purchased and kept overnight at roomtemperature, and then they were incubated in an incubator of 37° C. and70% humidity conditions. The dorsal aorta of the chicken embryo wasseparated on 14^(th) day of incubation. Clean aorta was obtained byremoving fat tissue surrounding the aorta, then were cut into size 1 mmusing blade. Matrigel (BD Bioscience) of 70 μl was spread onto a 96-wellplate and placed to congeal. Thereafter, the cut aorta were plantedthereto and covered by 50 μl of Matrigel. After congealing for 1 hour,100 μl volume of DMEM buffer, DMEM+VEGF (20 ng/ml), DMEM+VEGF (20ng/ml)+Non-CP-SOCS3 10 μM, DMEM+VEGF(20 ng/ml)+Avastin (1 mg/ml),DMEM+VEGF (20 ng/ml)+iCP-SOCS3(HM₁₆₅S3B) 10 μM was treated and weretaken pictures at 72 hours to observe sprouting.

As a result, it was investigated that the iCP-SOCS3 recombinant proteininhibited angiogenesis (FIG. 80).

Example 17-6. Investigation of Angiogenesis-Inhibitory Efficacy ofiCP-SOCS3 Recombinant Protein: CAM Assay

In order to investigate a mechanism-specific angiogenesis-inhibitoryefficacy of the iCP-SOCS3 recombinant protein, an in-vivo experimentalmodel was employed, and inhibition of angiogenesis required forembryonic development was investigated by a chick chorioallantoicmembrane (CAM) assay.

Fertile chicken eggs at 4 days after fertilization were placed in anincubator, and incubated at 34° C. and humidity of 50% for 24 hours. Asmall portion of eggshell above the air sac of the fertile chicken eggwas removed, and each 5 ml of albumin in the fertile chicken eggs wasremoved using a 10-ml syringe. After further incubation for 3 days, whenthe air sac was removed, angiogenesis was observed in the choriallantoicmembrane. The formed vessels were covered with cover glasses which werecoated with different concentrations of iCP-SOCS3 (0.5 μg, 2 μg), m),followed by incubation for 2 days. Angiogenesis at the region where thecover glass was placed was examined (FIG. 61). As a result, it wasinvestigated that the iCP-SOCS3 recombinant protein inhibitedangiogenesis in a concentration-dependent manner.

Example 18-1. Investigation of Tumor-Induced Angiogenesis-InhibitoryEfficacy of iCP-SOCS3 Recombinant Protein: Invasion Assay

Increased energy requirements of growing tumors cause activeangiogenesis in the peripheral tissues. To investigate whether migrationof vascular endothelial cells is blocked by inhibiting angiogenesisinducer secretion of tumors by iCP-SOCS3, inhibitory effects on humanumbilical vein endothelial cells (HUVECs) invasion were examined in atranswell using an iCP-SOCS3-treated tumor-conditioned medium.

First, PANC1 and U87MG cells were seeded at a density of 2×10⁵ in 6-wellplates, respectively. After 24 hours, the cells were treated with 10 μMof iCP-SOCS3 recombinant protein for 8 hours. HUVECs were seeded at adensity of 5×10⁴ in a transwell-coated with 0.1% gelatin, respectively.PANC-1 or U87MG conditioned media were filled into a bottom chamber ofthe transwell. The chamber and transwell were assembled, and 16 hourslater, the number of cells migrated to the lower side of the transwellwas counted using 0.1% crystal violet (FIG. 62). As a result, inhibitionrates of HUVEC migration by iCP-SOCS3 were 38% and 95%, respectively.

Example 18-2. Investigation of Tumor-Induced Angiogenesis-InhibitoryEfficacy of iCP-SOCS3 Recombinant Protein: Tumor Spheroid SproutingAssay

To investigate whether the angiogenesis-inhibitory efficacy of iCP-SOCS3affects growth rate and invasion of tumor, in-vitro conditions for tumorpreparation were determined. 100 ul of growth factor-free Matrigel wasadded to a 8-well chamber slide, followed by incubation at 30° C. forcoating. U87MG cells were seeded on the coated chamber slide at adensity of 1×10⁴, and cultured in a CO₂ incubator for 4 days, therebyU87MG spheroids were formed. The cells were treated with 10 uM ofiCP-SOCS3 recombinant protein for 4 days, and then the size of spheroidsand branches in each group were photographed using a microscope at 400×magnification. Two indices of spheroid size (FIG. 63) andinvasion-sprouting branch/length-(FIG. 64) were examined using a MultiGaugeV3.0 (FUJIFILM).

When HUVECs are added during spheroid formation, communication betweentwo cells is increased to develop spheroid. It was investigated thatfurther growth of spheroid did not occur by treatment of iCP-SOCS3recombinant protein.

Example 18-3. Investigation of Tumor-Induced Angiogenesis-InhibitoryEfficacy of iCP-SOCS3 Recombinant Protein: In-Vivo Matrigel Plug Assay

To investigate the angiogenesis-inhibitory efficacy of iCP-SOCS3recombinant protein, vascular migration in animals under actual tumorprogression was examined by a Matrigel plug assay.

Total of 28 Balb/C^(Nu/Nu) male mice (4-week-old) were prepared anddivided into a diluent group (n=6), a positive control group (n=4), anon-cp-SOCS3 group (n=8), and an iCP-SOCS3 (HM₁₆₅S3B) group (n=10). As atumor cell line for angiogenesis induction, U87MG cell was selected, and5×10⁶ thereof and 30 mg/kg of iCP-SOCS3 recombinant protein to betreated to each group were mixed with a Matrigel mixture. The positivecontrol was prepared by subcutaneous injection of a mixture of Matrigelwith 100 ng/ml of VEGF, 100 ng/ml of bFGF, and 100 U/ml of heparin intothe backs of mice, and observed for 3 weeks (FIG. 65). As a result, itwas investigated that no vascular formation was induced in theiCP-SOCS3-treated Matrigel plugs.

Proteins were isolated from tumor cells which were removed from Matrigelplugs. Then, each 20 μg of the proteins was loaded on a 12% SDS-PAGEgel, followed by electrophoresis at 125V for 1 hour and 30 minutes.Thereafter, the gel was transferred onto an NC membrane at 450 A for 1hour and 30 minutes. The membrane was then blocked with 5% non-fat milkin TBST, and incubated overnight at 4° C. with 1:2000 dilutions ofangiopoietin-2. Next day, the membrane was treated with 1:2000 dilutionof 2^(nd) antibody-conjugated HRP at RT for 1 hour. Protein expressionlevels of angiopoietin-2 were examined by an image analyzer. As aresult, it was confirmed that the expression levels ofangiogenesis-related factors were reduced in the iCP-SOCS3 recombinantprotein-treated groups (FIG. 81).

Example 19. Investigation of Tumor-Induced Angiogenesis InhibitionMechanism of iCP-SOCS3 Recombinant Protein

Female Balb/c^(nu/nu) mice were subcutaneously implanted with Hepaticcancer cell line (Hep3B, HepG2), pancreatic cancer cell line (PANC1),glioblastoma cell line (U87MG) tumor block (1 mm³) into the right backside of the mouse. Tumor-bearing mice were intravenously administeredwith iCP-SOCS3 or the control proteins (30 mg/kg) for 21 days andobserved for 2 or 3 weeks following the termination of the treatment.Tumor size was monitored by measuring the longest (length) and shortestdimensions (width) once a day with a dial caliper, and tumor volume wascalculated as width2×length×0.5.

Proteins were isolated from tumor tissues which were removed fromxenograft models. Then, each 20 ug of the proteins was loaded on a 12%SDS-PAGE gel, followed by electrophoresis at 125V for 1 hour and 30minutes. Thereafter, the gel was transferred onto an NC membrane at 450A for 1 hour and 30 minutes. The membrane was then blocked with 5%non-fat milk in TBST, and incubated overnight at 4° C. with 1:2000dilutions of VEGF and ERK/phosphor ERK. Next day, the membrane wastreated with 1:2000 dilution of 2^(nd) antibody-conjugated HRP at RT for1 hour. Protein expression levels of angiogenesis-related VEGF, ERK(phospho/total), Angiopoietin 1/2, and β-catenin (phospho/total) wereexamined by an image analyzer. As a result, it was confirmed that theexpression levels of angiogenesis-related factors were reduced in theiCP-SOCS3 recombinant protein-treated groups (Hep3b: FIG. 66, HepG2:FIG. 67, PANC1: FIG. 68, U87MG: FIG. 69).

The invention claimed is:
 1. A method of treating cancers or diseasesassociated with an angiogenic disorder in a subject in need thereof,comprising: administering to the subject a therapeutically effectiveamount of an improved Cell-Permeable (iCP)-SOCS3 recombinant protein,said iCP-SOCS3 recombinant protein comprising a SOCS3 protein and atleast one advanced macromolecule transduction domain (aMTD)(s) beingcomposed of 9-13 amino acid residues, wherein the aMTD is fused to oneend or both ends of the SOCS3 protein and has the following features(a)-(c): (a) being composed of 3 or more amino acid residuesindependently selected from the group consisting of Ala, Val, Ile, Leu,and Pro; (b) having Proline as an amino acid residue corresponding toany one or more of positions 5, 7 and 8, and position 12 of its aminoacid sequence; and (c) having an instability index of 40-60, analiphatic index of 180-220, and a grand average of hydropathy (GRAVY)score of 2.1-2.6, according to a Protparam computational formula,wherein the aMTD does not have proline at position
 6. 2. The methodaccording to claim 1, wherein the iCP-SOCS3 recombinant protein furthercomprises one or more solubilization domain (SD)(s), and the order ofaMTD(s), the SOCS3 protein and the SD(s) in the iCP-SOCS3 recombinantprotein are in any order.
 3. The method according to claim 1, whereinthe aMTD(s) is composed of 12 amino acid residues and represented by thefollowing general formula:

wherein, X(s) independently refer to Alanine (A), Valine (V), Leucine(L) or Isoleucine (I); Proline (P) is positioned in one of U(s) atposition 5, 7 or 8, and the remaining U(s) are independently composed ofA, V, L or I; and P at position 12 is Proline.
 4. The method accordingto claim 1, wherein the SOCS3 protein has the amino acid sequence of SEQID NO:
 814. 5. The method according to claim 4, wherein the SOCS3protein is encoded by the polynucleotide sequence of SEQ ID NO:
 815. 6.The method according to claim 1, wherein the at least one aMTD(s) has anamino acid sequence independently selected from the group consisting ofSEQ ID NOs: 2, 14-17, 22, 26, 28, 43-45, 60, 63, 68, 70, 71, 74-146,148-173, 175, 178-219, and 222-237.
 7. The method according to claim 6,wherein the at least one aMTD(s) is encoded by a polynucleotide sequenceindependently selected from the group consisting of SEQ ID NOs: 242,254-257, 262, 266, 268, 283-285, 300, 303, 308, 310, 311, 314-386,388-413, 415, 418-459 and 462-477.
 8. The method according to claim 2,wherein the one or more SD(s) has an amino acid sequence independentlyselected from the group consisting of SEQ ID NOs: 798 and
 799. 9. Themethod according to claim 8, wherein the one or more SD(s) is encoded bya polynucleotide sequence independently selected from the groupconsisting of SEQ ID NOs: 805 and
 806. 10. The method according to claim1, wherein the iCP-SOCS3 recombinant protein has a histidine-tagaffinity domain additionally fused to one end thereof.
 11. The methodaccording to claim 10, wherein the histidine-tag affinity domain has theamino acid sequence of SEQ ID NO:
 812. 12. The method according to claim11, wherein the histidine-tag affinity domain is encoded by thepolynucleotide sequence of SEQ ID NO: 813.